Data processing system for identifying a therapeutic agent

ABSTRACT

Provided herein are methods and systems of molecular profiling of diseases, such as cancer. In some embodiments, the molecular profiling can be used to identify treatments for the disease, such as treatments that provide likely benefit or likely lack of benefit for the disease.

CROSS-REFERENCE

This application is a Continuation of U.S. patent application Ser. No.16/675,488, filed on Nov. 6, 2019, which is a Continuation of U.S.patent application Ser. No. 15/555,378, filed on Sep. 1, 2017, which isa U.S. National Phase application under 35 U.S.C. 371 of InternationalPatent Application No. PCT/US2016/020657, which claims the benefit ofpriority to United States Provisional Patent Application Serial Nos.62/127,769, filed on Mar. 3, 2015, and 62/167,659, filed on May 28,2015; all of which applications are incorporated by reference herein intheir entirety.

BACKGROUND

Disease states in patients are typically treated with treatment regimensor therapies that are selected based on clinical based criteria; thatis, a treatment therapy or regimen is selected for a patient based onthe determination that the patient has been diagnosed with a particulardisease (which diagnosis has been made from classical diagnosticassays). Although the molecular mechanisms behind various disease stateshave been the subject of studies for years, the specific application ofa diseased individual's molecular profile in determining treatmentregimens and therapies for that individual has been disease specific andnot widely pursued.

Some treatment regimens have been determined using molecular profilingin combination with clinical characterization of a patient such asobservations made by a physician (such as a code from the InternationalClassification of Diseases, for example, and the dates such codes weredetermined), laboratory test results, x-rays, biopsy results, statementsmade by the patient, and any other medical information typically reliedupon by a physician to make a diagnosis in a specific disease. However,using a combination of selection material based on molecular profilingand clinical characterizations (such as the diagnosis of a particulartype of cancer) to determine a treatment regimen or therapy presents arisk that an effective treatment regimen may be overlooked for aparticular individual since some treatment regimens may work well fordifferent disease states even though they are associated with treating aparticular type of disease state.

Patients with refractory or metastatic cancer are of particular concernfor treating physicians. The majority of patients with metastatic orrefractory cancer eventually run out of treatment options or may suffera cancer type with no real treatment options. For example, some patientshave very limited options after their tumor has progressed in spite offront line, second line and sometimes third line and beyond) therapies.For these patients, molecular profiling of their cancer may provide theonly viable option for prolonging life.

More particularly, additional targets or specific therapeutic agents canbe identified assessment of a comprehensive number of targets ormolecular findings examining molecular mechanisms, genes, gene expressedproteins, and/or combinations of such in a patient's tumor. Identifyingmultiple agents that can treat multiple targets or underlying mechanismswould provide cancer patients with a viable therapeutic alternative on apersonalized basis so as to avoid standar therapies, which may simplynot work or identify therapies that would not otherwise be considered bythe treating physician.

There remains a need for better theranostic assessment of cancervictims, including molecular profiling analysis that identifies at leastone individual profile to provide more informed and effectivepersonalized treatment options, resulting in improved patient care andenhanced treatment outcomes. The present invention provides methods andsystems for identifying treatments for these individuals by molecularprofiling a sample from the individual. The molecular profiling caninclude analysis of immune modulators such as PD-1 and/or its ligandPD-L1.

SUMMARY OF THE INVENTION

The present invention provides methods and system for molecularprofiling, using the results from molecular profiling to identifytreatments for individuals. In some embodiments, the treatments were notidentified initially as a treatment for the disease or disease lineage.The molecular profiling can include analysis of a sequence of a nucleicacid. The sequence can be assessed in multiple aspects, e.g., for thepresence or absence of any detectable chromosomal or transcriptabnormality. Such a chromosomal or transcript abnormality may comprisewithout limitation a mutation, a polymorphism, a deletion, an insertion,a substitution, a translocation, a fusion, a break, a duplication, anamplification, a repeat, a copy number variant, a DNA methylationvariation, a transcript expression level, a transcript variant, and asplice variant.

In an aspect, the invention provides a method of identifying at leastone treatment associated with a cancer in a subject, comprising: a)determining a molecular profile for at least one sample from the subjectby assessing a plurality of genes and/or gene products; and b)identifying, based on the molecular profile, at least one of: i) atleast one treatment that is associated with benefit for treatment of thecancer; ii) at least one treatment that is associated with lack ofbenefit for treatment of the cancer; and iii) at least one treatmentassociated with a clinical trial. The plurality of genes and/or geneproducts can be chosen from amongst genes and or gene products (e.g.,transcripts and proteins) with efficacy known to be related to variouschemotherapeutic agents. In one non-limiting example, it may be knownthat an individual with a tumor that express a certain biomarker haslikely benefit of a given treatment whereas an individual with a tumorthat does not express that biomarker has likely lack of benefit of thetreatment. For example, HER2+ tumors may respond to the anti-HER2antibody whereas HER2− tumors would likely receive no benefit from suchtreatment. In another non-limiting example, a certain drug may havelikely benefit from a tumor carrying a wild type gene but not effectiveagainst a tumor carrying a given mutation in the same gene. For example,tumors with EGFR wild type may be treatable with an EGFR tyrosine kinaseinhibitor (TKI), such as gefitinib and erlotinib, whereas EGFR T790Mmutants are resistant to such treatments.

In an embodiment of the method of the invention, the cancer comprises abladder cancer and assessing the plurality of genes and/or gene productscomprises protein analysis of at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8or 9, of ERCC1, Her2/Neu, PD-L1, PTEN, RRM1, TOP2A, TOPO1, TS, TUBB3;and/or nucleic acid analysis of at least TOP2A.

In another embodiment of the method of the invention, the cancercomprises a breast cancer and assessing the plurality of genes and/orgene products comprises protein analysis of at least one, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or 11 of AR, ER, ERCC1, Her2/Neu, PD-L1, PR, PTEN,RRM1, TLE3, TOPO1, TS; and/or nucleic acid analysis of at least one ortwo of Her2/Neu and TOP2A.

In still another embodiment of the method of the invention, the cancercomprises a cancer of unknown primary (CUP) and assessing the pluralityof genes and/or gene products comprises protein analysis of at leastone, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of AR, ER, ERCC1,Her2/Neu, PD-L1, PR, PTEN, RRM1, TOP2A, TOPO1, TS, TUBB3; and/or nucleicacid analysis of at least Her2/Neu.

In yet embodiment of the method of the invention, the cancer comprises acervical cancer and assessing the plurality of genes and/or geneproducts comprises protein analysis of at least one, e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or 11 of ER, ERCC1, Her2/Neu, PD-L1, PR, PTEN, RRM1,TOP2A, TOPO1, TS, TUBB3; and/or nucleic acid analysis of at least one ortwo of Her2/Neu and TOP2A.

In an embodiment of the method of the invention, the cancer comprises acolorectal cancer (CRC) and assessing the plurality of genes and/or geneproducts comprises protein analysis of at least one, e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or 11 of ERCC1, HER2/Neu, MGMT, MLH1, MSH2, MSH6,PD-L1, PMS2, PTEN, TOPO1, TS; and/or nucleic acid analysis of at leastone or two of Her2/Neu and TOP2A; and/or MSI analysis.

In another embodiment of the method of the invention, the cancercomprises an endometrial cancer and assessing the plurality of genesand/or gene products comprises protein analysis of at least one, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of ER, ERCC1,Her2/Neu, MLH1, MSH2, MSH6, PD-L1, PMS2, PR, PTEN, RRM1, TOP2A, TOPO1,TS, TUBB3; and/or nucleic acid analysis of at least Her2/Neu; and/or MSIanalysis.

In still another embodiment of the method of the invention, the cancercomprises a gastric/esophageal cancer and assessing the plurality ofgenes and/or gene products comprises protein analysis of at least one,e.g., 1, 2, 3, 4, 5, 6, 7 or 8 of ERCC1, Her2/Neu, PD-L1, PTEN, TOP2A,TOPO1, TS, TUBB3; and/or nucleic acid analysis of at least Her2/Neu.

In yet another embodiment of the method of the invention, the cancercomprises a gastrointestinal stromal tumor (GIST) and assessing theplurality of genes and/or gene products comprises protein analysis of atleast one, e.g., 1, 2, 3 or 4 of ERCC1, Her2/Neu, PD-L1, PTEN; and/ornucleic acid analysis of at least Her2/Neu.

In an embodiment of the method of the invention, the cancer comprises aglioma and assessing the plurality of genes and/or gene productscomprises protein analysis of at least one, e.g., 1, 2, 3, 4, 5, 6 or 7of ERCC1, Her2/Neu, PD-L1, PTEN, TOPO1, TS, TUBB3; and/or nucleic acidanalysis of at least one or two of Her2/Neu and 1p19q; and/or fragmentanalysis of at least EGFR Variant III; and/or MGMT promoter methylationanalysis, e.g., by pyrosequencing.

In another embodiment of the method of the invention, the cancercomprises a head & neck cancer and assessing the plurality of genesand/or gene products comprises protein analysis of at least one, e.g.,1, 2, 3, 4, 5, 6 or 7 of ERCC1, Her2/Neu, PD-L1, PTEN, RRM1, TS, TUBB3;and/or nucleic acid analysis of at least Her2/Neu.

In yet another embodiment of the method of the invention, the cancercomprises a kidney cancer and assessing the plurality of genes and/orgene products comprises protein analysis of at least one, e.g., 1, 2, 3,4, 5, 6, 7, 8 or 9 of ERCC1, Her2/Neu, PD-L1, PTEN, RRM1, TOP2A, TOPO1,TS, TUBB3; and/or nucleic acid analysis of at least Her2/Neu.

In still another embodiment of the method of the invention, the cancercomprises a melanoma and assessing the plurality of genes and/or geneproducts comprises protein analysis of at least one, e.g., 1, 2, 3, 4,5, 6 or 7 of ERCC1, Her2/Neu, MGMT, PD-L1, PTEN, TS, TUBB3; and/ornucleic acid analysis of at least Her2/Neu.

In an embodiment of the method of the invention, the cancer comprises aa non-small cell lung cancer (NSCLC) and assessing the plurality ofgenes and/or gene products comprises protein analysis of at least one,e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 of ALK, ERCC1, Her2/Neu, PD-L1, PTEN,RRM1, TOPO1, TS, TUBB3; and/or nucleic acid analysis of at least one,e.g., 1, 2, 3 or 4 of cMET, EGFR, Her2/Neu and ROS1.

In another embodiment of the method of the invention, the cancercomprises an ovarian cancer and assessing the plurality of genes and/orgene products comprises protein analysis of at least one, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 of ER, ERCC1, Her2/Neu, PD-L1, PTEN, RRM1, TOP2A,TOPO1, TS, TUBB3; and/or nucleic acid analysis of at least Her2/Neu.

In yet another embodiment of the method of the invention, the cancercomprises a pancreatic/hepatobiliary/cholangiocarcinoma cancer andassessing the plurality of genes and/or gene products comprises proteinanalysis of at least one, e.g., 1, 2, 3, 4, 5, 6, 7 or 8 of ERCC1,Her2/Neu, PD-L1, PTEN, RRM1, TOPO1, TS, TUBB3; and/or nucleic acidanalysis of at least Her2/Neu.

In some embodiments of the method of the invention, the cancer comprisesa prostate cancer and assessing the plurality of genes and/or geneproducts comprises protein analysis of at least one, e.g., 1, 2, 3, 4,5, 6 or 7 of AR, ERCC1, Her2/Neu, PD-L1, PTEN, TOP2A, TUBB3; and/ornucleic acid analysis of at least Her2/Neu.

In an embodiment of the method of the invention, the cancer comprises asarcoma and assessing the plurality of genes and/or gene productscomprises protein analysis of at least one, e.g., 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 of ERCC1, Her2/Neu, MGMT, PD-L1, PTEN, RRM1, TOP2A, TOPO1,TS, TUBB3; and/or nucleic acid analysis of at least Her2/Neu.

In another embodiment of the method of the invention, the cancercomprises a thyroid cancer and assessing the plurality of genes and/orgene products comprises protein analysis of at least one, e.g., 1, 2, 3,4 or 5 of ERCC1, Her2/Neu, PD-L1, PTEN, TOP2A; and/or nucleic acidanalysis of at least Her2/Neu.

In still another embodiment of the method of the invention, the cancercomprises a solid tumor and assessing the plurality of genes and/or geneproducts comprises protein analysis of at least one, e.g., 1, 2, 3, 4,5, 6, 7 or 8 of ERCC1, Her2/Neu, PD-L1, PTEN, TOP2A, TOPO1, TS, TUBB3;and/or nucleic acid analysis of at least Her2/Neu.

Any useful laboratory method for protein analysis and/or nucleic acidanalysis can be used to carry out the methods of the invention. Forexample, proteins can be assessed using various forms of immunoassay, bymass based detection, or other techniques such as disclosed herein.Nucleic acids can be assessed by various amplification, hybridization,sequencing, or other techniques such as disclosed herein. In someembodiments, the protein analysis comprises immunohistochemistry (IHC)and/or the nucleic acid analysis comprises in situ hybridization (ISH).

The methods of the invention may further comprise mutational analysisperformed on any desired panel of genes. In an embodiment, assessing theplurality of genes and/or gene products further comprises mutationalanalysis of at least one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or46, of ABL1, AKT1, ALK, APC, ATM, BRAF, BRCA1, BRCA2, CDH1, CSF1R,CTNNB1, EGFR, ERBB2 (HER2), ERBB4 (HER4), FBXW7, FGFR1, FGFR2, FLT3,GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT(cKIT), KRAS, MET (cMET), MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN,PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53 and VHL. Themutational analysis may comprise any useful combination of these genes.

In another embodiment, assessing the plurality of genes and/or geneproducts further comprises using mutational analysis to assess at leastone, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57 or 58, of ABL1, AKT1, ALK, APC, AR, ARAF, ATM, BAP1,BRAF, BRCA1, BRCA2, CDK4, CDKN2A, CHEK1, CHEK2, CSF1R, CTNNB1, DDR2,EGFR, ERBB2, ERBB3, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HRAS,IDH1, IDH2, JAK2, KDR, KIT, KRAS, MAP2K1 (MEK1), MAP2K2 (MEK2), MET,MLH1, MPL, NF1, NOTCH1, NRAS, NTRK1, PDGFRA, PDGFRB, PIK3CA, PTCH1,PTEN, RAF1, RET, ROS1, SMO, SRC, TP53, VHL and WT1. The mutationalanalysis may comprise any useful selection or combination of thesegenes.

In still another embodiment, assessing the plurality of genes and/orgene products further comprises mutational analysis to assess at leastone gene, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or all, of the geneslisted in Table 12. The mutational analysis may comprise any usefulselection or combination of these genes.

In yet another embodiment, assessing the plurality of genes and/or geneproducts further comprises mutational analysis to assess at least one,e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, or all, of the genes listed in Table 13. The mutationalanalysis may comprise any useful selection or combination of thesegenes.

In an embodiment, assessing the plurality of genes and/or gene productsfurther comprises mutational analysis to assess at least one gene, e.g.,at least 1, 2, 3, 4, 5, 6, 7 or 8, of the genes listed in Table 14. Themutational analysis may comprise any useful combination of these genes.

In another embodiment, assessing the plurality of genes and/or geneproducts further comprises mutational analysis to assess at least one,e.g., at least 1 or 2, of the genes listed in Table 15 (EGFR vIII andMET Exon 14 Skipping). The mutational analysis may comprise any usefulselection or combination of these genes.

In still another embodiment, assessing the plurality of genes and/orgene products further comprises mutational analysis to assess at leastone, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250,300, 350, 400, 450, 500, 550, 600 or all, of the genes listed in Tables12-15, and any combination thereof. The mutational analysis may compriseany useful selection or combination of these genes.

In yet another embodiment, assessing the plurality of genes and/or geneproducts further comprises mutational analysis to assess at least one,e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, 450, 500, or all, of ABI1, ABL2, ACSL3, ACSL6, AFF1, AFF3,AFF4, AKAP9, AKT2, AKT3, ALDH2, AMER1, AR, ARFRP1, ARHGAP26, ARHGEF12,ARID1A, ARID2, ARNT, ASPSCR1, ASXL1, ATF1, ATIC, ATP1A1, ATP2B3, ATR,ATRX, AURKA, AURKB, AXIN1, AXL, BARD1, BCL10, BCL11A, BCL11B, BCL2,BCL2L11, BCL2L2, BCL3, BCL6, BCL7A, BCL9, BCOR, BCORL1, BCR, BIRC3, BLM,BMPR1A, BRD3, BRD4, BRIP1, BTG1, BTK, BUB1B, C11orf30, C15orf21,C15orf55, C15orf65, C16orf75, C2orf44, CACNA1D, CALR, CAMTA1, CANT1,CARD11, CARS, CASC5, CASP8, CBFA2T3, CBFB, CBL, CBLB, CBLC, CCDC6,CCNB1IP1, CCND1, CCND2, CCND3, CCNE1, CD274, CD74, CD79A, CD79B, CDC73,CDH11, CDK12, CDK4, CDK6, CDK8, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CDX2,CEBPA, CHCHD7, CHIC2, CHN1, CIC, CIITA, CLP1, CLTC, CLTCL1, CNBP, CNOT3,CNTRL, COL1A1, COPB1, COX6C, CREB1, CREB3L1, CREB3L2, CREBBP, CRKL,CRLF2, CRTC1, CRTC3, CSF3R, CTCF, CTLA4, CTNNA1, CXCR7, CYLD, CYP2D6,DAXX, DDB2, DDIT3, DDX10, DDX5, DDX6, DEK, DICER1, DNM2, DNMT3A, DOT1L,DUX4, EBF1, ECT2L, EIF4A2, ELF4, ELK4, ELL, ELN, EML4, EP300, EPHA3,EPHA5, EPHB1, EPS15, ERC1, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERG, ESR1,ETV1, ETV4, ETV5, ETV6, EWSR1, EXT1, EXT2, EZH2, EZR, FAM123B, FAM22A,FAM22B, FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, FAS,FBXO11, FCGR2B, FCRL4, FEV, FGF10, FGF14, FGF19, FGF23, FGF3, FGF4,FGF6, FGFR1OP, FGFR3, FGFR4, FH, FHIT, FIP1L1, FLCN, FLI1, FLT1, FLT4,FNBP1, FOXA1, FOXL2, FOXO1, FOXO3, FOXO4, FOXP1, FSTL3, FUBP1, FUS,GAS7, GATA1, GATA2, GATA3, GID4, GMPS, GNA13, GOLGA5, GOPC, GPC3, GPHN,GPR124, GRIN2A, GSK3B, H3F3A, H3F3B, HERPUD1, HEY1, HGF, HIP1, HIST1H3B,HIST1H4I, HLF, HMGA1, HMGA2, HNRNPA2B1, HOOK3, HOXA11, HOXA13, HOXA9,HOXC11, HOXC13, HOXD11, HOXD13, HSP90AA1, HSP90AB1, IGF1R, IKBKE, IKZF1,IL2, IL21R, IL6ST, IL7R, INHBA, IRF4, IRS2, ITK, JAK1, JAZF1, JUN,KAT6A, KCNJ5, KDM5A, KDM5C, KDM6A, KDSR, KEAP1, KIAA1549, KIF5B, KLF4,KLHL6, KLK2, KTN1, LASP1, LCK, LCP1, LGR5, LHFP, LIFR, LMO1, LMO2, LPP,LRIG3, LRP1B, LYL1, MAF, MAFB, MALT1, MAML2, MAP2K1 (MEK1), MAP2K2(MEK2), MAP2K4, MAP3K1, MAX, MCL1, MDM2, MDM4, MDS2, MECOM, MED12,MEF2B, MEN1, MITF, MKL1, MLF1, MLL, MLL2, MLL3, MLLT1, MLLT10, MLLT11,MLLT3, MLLT4, MLLT6, MN1, MNX1, MRE11A, MSH2, MSH6, MSI2, MSN, MTCP1,MTOR, MUC1, MUTYH, MYB, MYC, MYCL1, MYCN, MYD88, MYH11, MYH9, MYST4,NACA, NBN, NCKIPSD, NCOA1, NCOA2, NCOA4, NDRG1, NF2, NFE2L2, NFIB,NFKB2, NFKBIA, NIN, NKX2-1, NONO, NOTCH2, NR4A3, NSD1, NT5C2, NTRK2,NTRK3, NUMA1, NUP214, NUP93, NUP98, OLIG2, OMD, P2RY8, PAFAH1B2, PAK3,PALB2, PATZ1, PAX3, PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1, PCSK7, PDCD1,PDCD1LG2, PDE4DIP, PDGFB, PDGFRB, PDK1, PER1, PHF6, PHOX2B, PICALM,PIK3CG, PIK3R1, PIK3R2, PIM1, PLAG1, PML, PMS1, PMS2, POLE, POT1,POU2AF1, POU5F1, PPARG, PPP2R1A, PRCC, PRDM1, PRDM16, PRF1, PRKAR1A,PRKDC, PRRX1, PSIP1, PTCH1, PTPRC, RABEP1, RAC1, RAD21, RAD50, RAD51,RADSIL1, RALGDS, RANBP17, RAP1GDS1, RARA, RBM15, RECQL4, REL, RHOH,RICTOR, RNF213, RNF43, RPL10, RPL22, RPL5, RPN1, RPTOR, RUNDC2A, RUNX1,RUNx1T1, SBDS, SDC4, SDHAF2, SDHB, SDHC, SDHD, SEPT5, SEPT6, SEPT9, SET,SETBP1, SETD2, SF3B1, SFPQ, SFRS3, SH2B3, SH3GL1, SLC34A2, SLC45A3,SMAD2, SMARCA4, SMARCE1, SOCS1, SOX10, SOX2, SPECC1, SPEN, SPOP, SRC,SRGAP3, SRSF2, SS18, SS18L1, SSX1, SSX2, SSX4, STAG2, STAT3, STAT4,STATSB, STIL, SUFU, SUZ12, SYK, TAF15, TAL1, TAL2, TBL1XR1, TCEA1,TCF12, TCF3, TCF7L2, TCL1A, TERT, TET1, TET2, TFE3, TFEB, TFG, TFPT,TFRC, TGFBR2, THRAP3, TLX1, TLX3, TMPRSS2, TNFAIP3, TNFRSF14, TNFRSF17,TOP1, TPM3, TPM4, TPR, TRAF7, TRIM26, TRIM27, TRIM33, TRIP11, TRRAP,TSC1, TSC2, TSHR, TTL, U2AF1, UBR5, USP6, VEGFA, VEGFB, VTI1A, WAS,WHSC1, WHSC1L1, WIF1, WISP3, WRN, WWTR1, XPA, XPC, XPO1, YWHAE, ZBTB16,ZMYM2, ZNF217, ZNF331, ZNF384, ZNF521, ZNF703 and ZRSR2. The mutationalanalysis may comprise any useful selection or combination of thesegenes.

In an embodiment, assessing the plurality of genes and/or gene productsfurther comprises using mutational analysis to assess at least one,e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, 450, 500, 550, or all, of ABI1, ABL1, ABL2, ACKR3, ACSL3,ACSL6, AFF1, AFF3, AFF4, AKAP9, AKT1, AKT2, AKT3, ALDH2, ALK, AMER1(FAM123B), APC, AR, ARAF, ARFRP1, ARHGAP26, ARHGEF12, ARID1A, ARID2,ARNT, ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATP1A1, ATP2B3, ATR, ATRX, AURKA,AURKB, AXIN1, AXL, BAP1, BARD1, BCL10, BCL11A, BCL11B, BCL2, BCL2L11,BCL2L2, BCL3, BCL6, BCL7A, BCL9, BCOR, BCORL1, BCR, BIRC3, BLM, BMPR1A,BRAF, BRCA1, BRCA2, BRD3, BRD4, BRIP1, BTG1, BTK, BUB1B, C11orf30(EMSY), C15orf65, C2orf44, CACNA1D, CALR, CAMTA1, CANT1, CARD11, CARS,CASC5, CASP8, CBFA2T3, CBFB, CBL, CBLB, CBLC, CCDC6, CCNB1IP1, CCND1,CCND2, CCND3, CCNE1, CD274 (PDL1), CD74, CD79A, CD79B, CDC73, CDH1,CDH11, CDK12, CDK4, CDK6, CDK8, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CDX2,CEBPA, CHCHD7, CHEK1, CHEK2, CHIC2, CHN1, CIC, CIITA, CLP1, CLTC,CLTCL1, CNBP, CNOT3, CNTRL, COL1A1, COPB1, COX6C, CREB1, CREB3L1,CREB3L2, CREBBP, CRKL, CRLF2, CRTC1, CRTC3, CSF1R, CSF3R, CTCF, CTLA4,CTNNA1, CTNNB1, CYLD, CYP2D6, DAXX, DDB2, DDIT3, DDR2, DDX10, DDX5,DDX6, DEK, DICER1, DNM2, DNMT3A, DOT1L, EBF1, ECT2L, EGFR, EIF4A2, ELF4,ELK4, ELL, ELN, EML4, EP300, EPHA3, EPHA5, EPHB1, EPS15, ERBB2 (HER2),ERBB3 (HER3), ERBB4 (HER4), ERC1, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5,ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EWSR1, EXT1, EXT2, EZH2, EZR, FAM46C,FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, FAS, FBX011, FBXW7,FCRL4, FEV, FGF10, FGF14, FGF19, FGF23, FGF3, FGF4, FGF6, FGFR1,FGFR1OP, FGFR2, FGFR3, FGFR4, FH, FHIT, FIP1L1, FLCN, FLI1, FLT1, FLT3,FLT4, FNBP1, FOXA1, FOXL2, FOXO1, FOXO3, FOXO4, FOXP1, FSTL3, FUBP1,FUS, GAS7, GATA1, GATA2, GATA3, GID4 (C17orf39), GMPS, GNA11, GNA13,GNAQ, GNAS, GOLGA5, GOPC, GPC3, GPHN, GPR124, GRIN2A, GSK3B, H3F3A,H3F3B, HERPUD1, HEY1, HGF, HIP1, HIST1H3B, HIST1H4I, HLF, HMGA1, HMGA2,HMGN2P46, HNF1A, HNRNPA2B1, HOOK3, HOXA11, HOXA13, HOXA9, HOXC11,HOXC13, HOXD11, HOXD13, HRAS, HSP9OAA1, HSP90AB1, IDH1, IDH2, IGF1R,IKBKE, IKZFL IL2, IL21R, IL6ST, IL7R, INHBA, IRF4, IRS2, ITK, JAK1,JAK2, JAK3, JAZF1, JUN, KAT6A (MYST3), KAT6B, KCNJ5, KDM5A, KDM5C,KDM6A, KDR, KDSR, KEAP1, KIAA1549, KIF5B, KIT, KLF4, KLHL6, KLK2, KMT2A(MLL), KMT2C (MLL3), KMT2D (MLL2), KRAS, KTN1, LASP1, LCK, LCP1, LGR5,LHFP, LIFR, LMO1, LMO2, LPP, LRIG3, LRP1B, LYL1, MAF, MAFB, MALT1,MAML2, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAX, MCL1, MDM2, MDM4, MDS2,MECOM, MED12, MEF2B, MEN1, MET, MITF, MKL1, MLF1, MLH1, MLLT1, MLLT10,MLLT11, MLLT3, MLLT4, MLLT6, MN1, MNX1, MPL, MRE11A, MSH2, MSH6, MSI2,MSN, MTCP1, MTOR, MUC1, MUTYH, MYB, MYC, MYCL (MYCL1), MYCN, MYD88,MYH11, MYH9, NACA, NBN, NCKIPSD, NCOA1, NCOA2, NCOA4, NDRG1, NF1, NF2,NFE2L2, NFIB, NFKB2, NFKBIA, NIN, NKX2-1, NONO, NOTCH1, NOTCH2, NPM1,NR4A3, NRAS, NSD1, NT5C2, NTRK1, NTRK2, NTRK3, NUMA1, NUP214, NUP93,NUP98, NUTM1, NUTM2B, OLIG2, OMD, P2RY8, PAFAH1B2, PAK3, PALB2, PATZ1,PAX3, PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1, PCSK7, PDCD1 (PD1), PDCD1LG2(PDL2), PDE4DIP, PDGFB, PDGFRA, PDGFRB, PDK1, PER1, PHF6, PHOX2B,PICALM, PIK3CA, PIK3CG, PIK3R1, PIK3R2, PIM1, PLAG1, PML, PMS1, PMS2,POLE, POT1, POU2AF1, POU5F1, PPARG, PPP2R1A, PRCC, PRDM1, PRDM16, PRF1,PRKAR1A, PRKDC, PRRX1, PSIP1, PTCH1, PTEN, PTPN11, PTPRC, RABEP1, RAC1,RAD21, RAD50, RAD51, RAD51B, RAF1, RALGDS, RANBP17, RAP1GDS1, RARA, RB1,RBM15, RECQL4, REL, RET, RHOH, RICTOR, RMI2, RNF213, RNF43, ROS1, RPL10,RPL22, RPL5, RPN1, RPTOR, RSPO3, RUNX1, RUNx1T1, SBDS, SDC4, SDHAF2,SDHB, SDHC, SDHD, SEPT5, SEPT6, SEPT9, SET, SETBP1, SETD2, SF3B1, SFPQ,SH2B3, SH3GL1, SLC34A2, SLC45A3, SMAD2, SMAD4, SMARCA4, SMARCB1,SMARCE1, SMO, SNX29, SOCS1, SOX10, SOX2, SPECC1, SPEN, SPOP, SRC,SRGAP3, SRSF2, SRSF3, SS18, SS18L1, SSX1, STAG2, STAT3, STAT4, STATSB,STIL, STK11, SUFU, SUZ12, SYK, TAF15, TAL1, TAL2, TBL1XR1, TCEA1, TCF12,TCF3, TCF7L2, TCL1A, TERT, TET1, TET2, TFE3, TFEB, TFG, TFPT, TFRC,TGFBR2, THRAP3, TLX1, TLX3, TMPRSS2, TNFAIP3, TNFRSF14, TNFRSF17, TOP1,TP53, TPM3, TPM4, TPR, TRAF7, TRIM26, TRIM27, TRIM33, TRIP11, TRRAP,TSC1, TSC2, TSHR, TTL, U2AF1, UBR5, USP6, VEGFA, VEGFB, VHL, VTI1A, WAS,WHSC1, WHSC1L1, WIF1, WISP3, WRN, WT1, WWTR1, XPA, XPC, XPO1, YWHAE,ZBTB16, ZMYM2, ZNF217, ZNF331, ZNF384, ZNF521, ZNF703 and ZRSR2. Themutational analysis may comprise any useful selection or combination ofthese genes.

In another embodiment, assessing the plurality of genes and/or geneproducts further comprises using mutational analysis to assess at leastone, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750 or all, of ABCB1,ABCG2, ABI1, ABL1, ABL2, ACKR3, ACSL3, ACSL6, ACVR1B, ACVR2A, AFF1,AFF3, AFF4, AKAP9, AKT1, AKT2, AKT3, ALDH1A1, ALDH2, ALK, AMER1, ANGPT1,ANGPT2, ANKRD23, APC, AR, ARAF, AREG, ARFRP1, ARHGAP26, ARHGEF12,ARID1A, ARID1B, ARID2, ARNT, ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATP1A1,ATP2B3, ATR, ATRX, AURKA, AURKB, AXINL AXL, BAP1, BARD1, BBC3, BCL10,BCL11A, BCL11B, BCL2, BCL2L1, BCL2L11, BCL2L2, BCL3, BCL6, BCL7A, BCL9,BCOR, BCORL1, BCR, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD3, BRD4,BRINP3, BRIP1, BTG1, BTG2, BTK, BUB1B, C11orf30, C15orf65, C2orf44, CA6,CACNA1D, CALR, CAMTA1, CANT1, CARD11, CARS, CASC5, CASP8, CBFA2T3, CBFB,CBL, CBLB, CBLC, CCDC6, CCNB1IP1, CCND1, CCND2, CCND3, CCNE1, CD19,CD22, CD274, CD38, CD4, CD70, CD74, CD79A, CD79B, CD83, CDC73, CDH1,CDH11, CDK12, CDK4, CDK6, CDK7, CDK8, CDK9, CDKN1A, CDKN1B, CDKN2A,CDKN2B, CDKN2C, CDX2, CEBPA, CHCHD7, CHD2, CHD4, CHEK1, CHEK2, CHIC2,CHN1, CHORDC1, CIC, CIITA, CLP1, CLTC, CLTCL1, CNBP, CNOT3, CNTRL,COL1A1, COPB1, COX6C, CRBN, CREB1, CREB3L1, CREB3L2, CREBBP, CRKL,CRLF2, CRTC1, CRTC3, CSF1R, CSF3R, CTCF, CTLA4, CTNNA1, CTNNB1, CUL3,CXCR4, CYLD, CYP17A1, CYP2D6, DAXX, DDB2, DDIT3, DDR1, DDR2, DDX10,DDX3X, DDX5, DDX6, DEK, DICER1, DIS3, DLL4, DNM2, DNMT1, DNMT3A, DOT1L,DPYD, DUSP4, DUSP6, EBF1, ECT2L, EDNRB, EED, EGFR, EIF4A2, ELF4, ELK4,ELL, ELN, EML4, EP300, EPHA3, EPHA5, EPHA7, EPHA8, EPHB1, EPHB2, EPHB4,EPS15, ERBB2, ERBB3, ERBB4, ERC1, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5,EREG, ERG, ERN1, ERRFI1, ESR1, ETV1, ETV4, ETV5, ETV6, EWSR1, EXT1,EXT2, EZH2, EZR, FAF1, FAIM3, FAM46C, FANCA, FANCC, FANCD2, FANCE,FANCF, FANCG, FANCL, FAS, FAT1, FBXO11, FBXW7, FCRL4, FEV, FGF10, FGF14,FGF19, FGF2, FGF23, FGF3, FGF4, FGF6, FGFR1, FGFR1OP, FGFR2, FGFR3,FGFR4, FH, FHIT, FIP1L1, FKBP1A, FLCN, FLI1, FLT1, FLT3, FLT4, FNBP1,FOXA1, FOXL2, FOXO1, FOXO3, FOXO4, FOXP1, FRS2, FSTL3, FUBP1, FUS,GABRA6, GAS7, GATA1, GATA2, GATA3, GATA4, GATA6, GID4, GLI1, GMPS,GNA11, GNA12, GNA13, GNAQ, GNAS, GNRH1, GOLGA5, GOPC, GPC3, GPHN,GPR124, GRIN2A, GRM3, GSK3B, GUCY2C, H3F3A, H3F3B, HCK, HDAC1, HERPUD1,HEY1, HGF, HIP1, HIST1H1E, HIST1H3B, HIST1H4I, HLF, HMGA1, HMGA2,HMGN2P46, HNF1A, HNMT, HNRNPA2B1, HNRNPK, HOOK3, HOXA11, HOXA13, HOXA9,HOXC11, HOXC13, HOXD11, HOXD13, HRAS, HSD3B1, HSP90AA1, HSP90AB1, IAPP,ID3, IDH1, IDH2, IGF1R, IGF2, IKBKE, IKZF1, IL2, IL21R, IL3RA, IL6,IL6ST, IL7R, INHBA, INPP4B, IRF2, IRF4, IRS2, ITGAV, ITGB1, ITK, ITPKB,JAK1, JAK2, JAK3, JAZF1, JUN, KAT6A, KAT6B, KCNJ5, KDM1A, KDM5A, KDM5C,KDM6A, KDR, KDSR, KEAP1, KEL, KIAA1549, KIF5B, KIR3DL1, KIT, KLF4,KLHL6, KLK2, KMT2A, KMT2C, KMT2D, KRAS, KTN1, LASP1, LCK, LCP1, LGALS3,LGR5, LHFP, LIFR, LMO1, LMO2, LOXL2, LPP, LRIG3, LRP1B, LUC7L2, LYL1,LYN, LZTR1, MAF, MAFB, MAGED1, MAGI2, MALT1, MAML2, MAP2K1, MAP2K2,MAP2K4, MAP3K1, MAPK1, MAPK11, MAX, MCL1, MDM2, MDM4, MDS2, MECOM,MED12, MEF2B, MEN1, MET, MITF, MKI67, MKL1, MLF1, MLH1, MLLT1, MLLT10,MLLT11, MLLT3, MLLT4, MLLT6, MMP9, MN1, MNX1, MPL, MRE11A, MS4A1, MSH2,MSH6, MSI2, MSN, MST1R, MTCP1, MTF2, MTOR, MUC1, MUC16, MUTYH, MYB, MYC,MYCL, MYCN, MYD88, MYH11, MYH9, NACA, NAE1, NBN, NCKIPSD, NCOA1, NCOA2,NCOA4, NDRG1, NF1, NF2, NFE2L2, NFIB, NFKB2, NFKBIA, NIN, NKX2-1, NONO,NOTCH1, NOTCH2, NOTCH3, NPM1, NR4A3, NRAS, NSD1, NT5C2, NTRK1, NTRK2,NTRK3, NUMA1, NUP214, NUP93, NUP98, NUTM1, NUTM2B, OLIG2, OMD, P2RY8,PAFAH1B2, PAK3, PALB2, PARK2, PARP1, PATZ1, PAX3, PAX5, PAX7, PAX8,PBRM1, PBX1, PCM1, PCSK7, PDCD1, PDCD1LG2, PDE4DIP, PDGFB, PDGFRA,PDGFRB, PDK1, PECAM1, PER1, PHF6, PHOX2B, PICALM, PIK3C2B, PIK3CA,PIK3CB, PIK3CD, PIK3CG, PIK3R1, PIK3R2, PIM1, PLAG1, PLCG2, PML, PMS1,PMS2, POLD1, POLE, POT1, POU2AF1, POU5F1, PPARG, PPP2R1A, PRCC, PRDM1,PRDM16, PREX2, PRF1, PRKAR1A, PRKCI, PRKDC, PRLR, PRPF40B, PRRT2, PRRX1,PRSS8, PSIP1, PSMD4, PTBP1, PTCH1, PTEN, PTK2, PTPN11, PTPRC, PTPRD,QKI, RABEP1, RAC1, RAD21, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAF1,RALGDS, RANBP17, RANBP2, RAP1GDS1, RARA, RB1, RBM10, RBM15, RCOR1,RECQL4, REL, RELN, RET, RHOA, RHOH, RICTOR, RIPKL RMI2, RNF213, RNF43,ROS1, RPL10, RPL22, RPL5, RPN1, RPS6KB1, RPTOR, RUNX1, RUNX1T1, S1PR2,SAMHD1, SBDS, SDC4, SDHA, SDHAF2, SDHB, SDHC, SDHD, SEPT5, SEPT6, SEPT9,SET, SETBP1, SETD2, SF1, SF3A1, SF3B1, SF3B2, SFPQ, SGK1, SH2B3, SH3GL1,SLAMF7, SLC34A2, SLC45A3, SLIT2, SMAD2, SMAD3, SMAD4, SMARCA4, SMARCB1,SMARCE1, SMC1A, SMC3, SMO, SNCAIP, SNX29, SOCS1, SOX10, SOX11, SOX2,SOX9, SPECC1, SPEN, SPOP, SPTA1, SRC, SRGAP3, SRSF2, SRSF3, SS18,SS18L1, SSX1, STAG2, STAT3, STAT4, STATSB, STEAP1, STIL, STK11, SUFU,SUZ12, SYK, TAF1, TAF15, TAL1, TAL2, TBL1XR1, TBX3, TCEA1, TCF12, TCF3,TCF7L2, TCL1A, TEK, TERC, TERT, TET1, TET2, TFE3, TFEB, TFG, TFPT, TFRC,TGFB1, TGFBR2, THRAP3, TIMP1, TJP1, TLX1, TLX3, TM7SF2, TMPRSS2,TNFAIP3, TNFRSF14, TNFRSF17, TNFRSF18, TNFRSF9, TNFSF11, TOP1, TOP2A,TP53, TP63, TPBG, TPM3, TPM4, TPR, TRAF2, TRAF3, TRAF3IP3, TRAF7,TRIM26, TRIM27, TRIM33, TRIP11, TRRAP, TSC1, TSC2, TSHR, TTK, TTL, TYMS,U2AF1, U2AF2, UBA1, UBR5, USP6, VEGFA, VEGFB, VHL, VPS51, VTI1A, WAS,WEE1, WHSC1, WHSC1L1, WIF1, WISP3, WNT11, WNT2B, WNT3, WNT3A, WNT4,WNTSA, WNT6, WNT7B, WRN, WT1, WWTR1, XBP1, XPA, XPC, XPO1, YWHAE, YWHAZ,ZAK, ZBTB16, ZBTB2, ZMYM2, ZMYM3, ZNF217, ZNF331, ZNF384, ZNF521, ZNF703and ZRSR2. The mutational analysis may comprise any useful selection orcombination of these genes.

In still another embodiment, assessing the plurality of genes and/orgene products further comprises using mutational analysis to assess acopy number variation in at least one, e.g., at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or all, of ABL1, AKT1, AKT2,ALK, ANG1/ANGPT1/TM7SF2, ANG2/ANGPT2NPS51, APC, ARAF, ARID1A, ATM,AURKA, AURKB, BBC3, BCL2, BIRC3, BRAF, BRCA1, BRCA2, CCND1, CCND3,CCNE1, CDK4, CDK6, CDK8, CDKN2A, CHEK1, CHEK2, CREBBP, CRKL, CSF1R,CTLA4, CTNNB1, DDR2, EGFR, EP300, ERBB3, ERBB4, EZH2, FBXW7, FGF10,FGF3, FGF4, FGFR1, FGFR2, FGFR3, FLT3, GATA3, GNA11, GNAQ, GNAS, HNF1A,HRAS, IDH1, IDH2, JAK2, JAK3, KRAS, MCL1, MDM2, MLH1, MPL, MYC, NF1,NF2, NFKBIA, NOTCH1, NPM1, NRAS, NTRK1, PAX3, PAX5, PAX7, PAX8, PDGFRA,PDGFRB, PIK3CA, PTCH1, PTEN, PTPN11, RAF1, RB1, RET, RICTOR, ROS1,SMAD4, SRC, TOP1, TOP2A, TP53, VHL and WT1. The mutational analysis maycomprise any selection or useful combination of these genes.

In yet another embodiment, assessing the plurality of genes and/or geneproducts further comprises using mutational analysis to assess a genefusion in at least one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28or 29, of ALK, AR, BCR, BRAF, ETV1, ETV4, ETV5, ETV6, EWSR1, FGFR1,FGFR2, FGFR3, FUS, MYB, NFIB, NR4A3, NTRK1, NTRK2, NTRK3, PDGFRA, RAF1,RARA, RET, ROS1, SSX1, SSX2, SSX4, TFE3 and TMPRSS2. The mutationalanalysis may comprise any useful selection or combination of thesegenes.

In an embodiment, assessing the plurality of genes and/or gene productsfurther comprises using mutational analysis to assess a gene fusion inat least one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52 or 53, of AKT3, ALK, ARHGAP26, AXL, BRAF, BRD3, BRD4, EGFR,ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EWSR1, FGFR1, FGFR2, FGFR3, FGR,INSR, MAML2, MAST1, MAST2, MET, MSMB, MUSK, MYB, NOTCH1, NOTCH2, NRG1,NTRK1, NTRK2, NTRK3, NUMBL, NUTM1, PDGFRA, PDGFRB, PIK3CA, PKN1, PPARG,PRKCA, PRKCB, RAF1, RELA, RET, ROS1, RSPO2, RSPO3, TERT, TFE3, TFEB,THADA and TMPRSS2. The mutational analysis may comprise any usefulselection or combination of these genes.

In another embodiment, assessing the plurality of genes and/or geneproducts further comprises using mutational analysis to assess a genefusion in at least one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26, ofALK, CAMTA1, CCNB3, CIC, EPC, EWSR1, FKHR, FUS, GLI1, HMGA2, JAZF1,MEAF6, MKL2, NCOA2, NTRK3, PDGFB, PLAG1, ROS1, SS18, STATE, TAF15,TCF12, TFE3, TFG, USP6 and YWHAE.

In still another embodiment, assessing the plurality of genes and/orgene products further comprises using mutational analysis to assess agene fusion in at least one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or 12, of ABL1, ABL2, CSF1R, PDGFRB, CRLF2, JAK2, EPOR, IL2RB,NTRK3, PTK2B, TSLP and TYK2. The mutational analysis may comprise anyuseful selection or combination of these genes.

The mutational analysis can be used to assess at least one, e.g., atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 of a mutation, apolymorphism, a deletion, an insertion, a substitution, a translocation,a fusion, a break, a duplication, an amplification, a repeat, a copynumber variation, a transcript variant, and a splice variant. Themutational analysis can be performed using any useful laboratory methodor combination of methods. For example, the mutational analysis can beperformed using at least one of ISH, amplification, PCR, RT-PCR,hybridization, microarray, sequencing, pyrosequencing, Sangersequencing, high throughput or Next Generation sequencing (NGS),fragment analysis or RFLP. Other useful methods are disclosed herein. Insome embodiments, the mutational analysis comprises Next GenerationSequencing.

Additional genes or gene products can be assessed as desired. Forexample, additional genes of theranostic or prognostic benefit may bechosen to be assessed. The plurality of genes and/or gene productsfurther comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9, ofCAIX, hENT1, IDO, LAG3, RET, NTRK1 (NTRK, TRK), PD-1, H3K36me3 andPBRM1, and any combination thereof. H3K36me3 and PBRM1 may be assessedin the case of kidney cancer. The plurality of genes and/or geneproducts can be according to any one or more of Tables 7, 8, 12, 13, 14and 15.

As noted, any useful combination of laboratory techniques may be used todetermine the molecular profile.

In the methods of the invention, the step of identifying based on themolecular profile may comprise correlating the molecular profile withtreatments whose benefit has been assessed for cancers characterized bypresence or level, overexpression, underexpression, copy number,mutation, deletion, insertion, translocation, amplification,rearrangement, or other molecular alteration in at least one member ofthe plurality of gene or gene products. In some embodiments, the step ofcorrelating the molecular profile with treatments is according to atleast one biomarker-drug association in any of Tables 3-6, Tables 9-10,Table 17, and Tables 22-24.

Exemplary biomarker-drug association rules include the following: a)performing IHC on PD1 to determine likely benefit or lack of benefitfrom a PD-1 modulating therapy, PD-1 inhibitor, anti-PD-1 immunotherapy,anti-PD-1 monoclonal antibody, nivolumab, pidilizumab (CT-011, CureTech,LTD), pembrolizumab (lambrolizumab, MK-3475, Merck), a PD-1 antagonist,a PD-1 ligand soluble construct, and/or AMP-224 (Amplimmune); b)performing IHC on PD-L1 to determine likely benefit or lack of benefitfrom a PD-L1 modulating therapy, PD-L1 inhibitor, anti-PD-L1immunotherapy, anti-PD-L1 monoclonal antibody, BMS-936559,MPDL3280A/RG7446, and/or MEDI4736 (MedImmune); c) performing IHC on RRM1to determine likely benefit or lack of benefit from an antimetaboliteand/or gemcitabine; d) performing IHC on TS to determine likely benefitor lack of benefit from a antimetabolite, fluorouracil, capecitabine,and/or pemetrexed; e) performing IHC on TOPO1 to determine likelybenefit or lack of benefit from a TOPO1 inhibitor, irinotecan and/ortopotecan; f) performing at least one of IHC on MGMT, pyrosequencing forMGMT promoter methylation, and sequencing on IDH1 to determine likelybenefit or lack of benefit from an alkylating agent, temozolomide,and/or dacarbazine; g) performing IHC on AR to determine likely benefitor lack of benefit from an anti-androgen, bicalutamide, flutamide,abiraterone and/or enzalutamide; h) performing IHC on ER to determinelikely benefit or lack of benefit from a hormonal agent, tamoxifen,fulvestrant, letrozole, and/or anastrozole; i) performing IHC on atleast one of ER, PR and AR to determine likely benefit or lack ofbenefit from a hormonal agent, tamoxifen, toremifene, fulvestrant,letrozole, anastrozole, exemestane, megestrol acetate, leuprolide,goserelin, bicalutamide, flutamide, abiraterone, enzalutamide,triptorelin, abarelix, and/or degarelix; j) performing at least one ofIHC on HER2 and ISH on HER2 to determine likely benefit or lack ofbenefit from a tyrosine kinase inhibitor and/or lapatinib, pertuzumab,and/or ado-trastuzumab emtansine (T-DM1); k) performing at least one ofIHC on HER2, ISH on HER2, IHC on PTEN and sequencing on PIK3CA todetermine likely benefit or lack of benefit from HER2 targeted therapy,and/or trastuzumab; 1) performing at least one of ISH on TOP2A, ISH onHER2, IHC on TOP2A and IHC on PGP to determine likely benefit or lack ofbenefit from an anthracycline, doxorubicin, liposomal-doxorubicin,and/or epirubicin; m) performing sequencing on at least one of cKIT andPDGFRA to determine likely benefit or lack of benefit from a tyrosinekinase inhibitor and/or imatinib; n) performing at least one of ISH onALK and ISH on ROS1 to determine likely benefit or lack of benefit froma tyrosine kinase inhibitor and/or crizotinib; o) performing at leastone of IHC on ER or sequencing on PIK3CA to determine likely benefit orlack of benefit from an mTOR inhibitor, everolimus, and/or temsirolimus;p) performing sequencing on RET to determine likely benefit or lack ofbenefit from a tyrosine kinase inhibitor, and/or vandetanib; q)performing IHC on at least one of TLE3, TUBB3 and PGP to determinelikely benefit or lack of benefit from a taxane, paclitaxel, and/ordocetaxel; r) performing IHC on SPARC to determine likely benefit orlack of benefit from a taxane, and/or nab-paclitaxel; s) performing atleast one of PCR and sequencing on BRAF to determine likely benefit orlack of benefit from a tyrosine kinase inhibitor, vemurafenib,dabrafenib, and/or trametinib; t) performing at least one of sequencingon KRAS, sequencing on BRAF, sequencing on NRAS, sequencing on PIK3CAand IHC on PTEN to determine likely benefit or lack of benefit from anEGFR-targeted antibody, cetuximab, and/or panitumumab; u) performingsequencing on EGFR to determine likely benefit or lack of benefit froman EGFR-targeted antibody, and/or cetuximab; v) performing at least oneof sequencing on EGFR, sequencing on KRAS, ISH on cMET, sequencing onPIK3CA and IHC on PTEN to determine likely benefit or lack of benefitfrom a tyrosine kinase inhibitor, erlotinib, and/or gefitinib; w)performing sequencing on EGFR to determine likely benefit or lack ofbenefit from a tyrosine kinase inhibitor, and/or afatinib; x) performingsequencing on cKIT to determine likely benefit or lack of benefit from atyrosine kinase inhibitor, and/or sunitinib; y) performing sequencing onat least one of BRCA1, BRCA2 and/or IHC on ERCC1 to determine likelybenefit or lack of benefit from carboplatin, cisplatin, and/oroxaliplatin; z) performing ISH on ALK to determine likely benefit orlack of benefit from ceritinib; and aa) performing ISH to detect 1p19qcodeletion to determine likely benefit or lack of benefit fromprocarbazine, lomustine, and/or vincristine (PCV).

Any useful methodology can be used to determine biomarker-drugassociation rules. In an embodiment, the step of correlating themolecular profile with treatments is according to at least onebiomarker-drug association rule derived from review of the scientificliterature, data obtained from clinical trials, and/or from previousmolecular profiling results in individuals with similar cancers.

The methods of the invention may further comprise identifying at leastone candidate clinical trial for the subject based on the molecularprofiling.

Any useful biological sample can be used to carry out the methods of theinvention. In some embodiments, the sample comprises formalin-fixedparaffin-embedded (FFPE) tissue, fixed tissue, core needle biopsy, fineneedle aspirate, unstained slides, fresh frozen (FF) tissue, formalinsamples, tissue comprised in a solution that preserves nucleic acid orprotein molecules, a fresh sample, malignant fluid, and/or a bodilyfluid sample. Multiple samples and/or sample types can be assessed asdesired. The sample may comprise cells from a solid tumor. The samplemay also comprise a bodily fluid. The bodily fluid may comprise amalignant fluid. The bodily fluid may comprise a pleural fluid orperitoneal fluid. In some embodiments, the bodily fluid comprisesperipheral blood, sera, plasma, ascites, urine, cerebrospinal fluid(CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor,amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid,semen, prostatic fluid, cowper's fluid, pre-ejaculatory fluid, femaleejaculate, sweat, fecal matter, tears, cyst fluid, pleural fluid,peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile,interstitial fluid, menses, pus, sebum, vomit, vaginal secretions,mucosal secretion, stool water, pancreatic juice, lavage fluids fromsinus cavities, bronchopulmonary aspirates, blastocyst cavity fluid, orumbilical cord blood.

The at least one sample may comprise a microvesicle population. In suchcases, at least one member of the plurality of genes and/or geneproducts can be associated with the microvesicle population.

As described herein, molecular profiling of the invention may beperformed at any useful time in the course of treatment. For example,the molecular profiling may be performed before any treatment or anychemotherapy has been administered to the individual for the cancer. Themolecular profiling may also be performed after one or more priorchemotherapeutic regimen has been administered to the individual for thecancer. Such prior treatments may have failed. Molecular profiling maybe performed in the salvage treatment setting. The cancer may comprise ametastatic and/or recurrent cancer. The cancer may be refractory to aprior treatment. In some embodiments, the prior treatment comprises thestandard of care for the cancer. The cancer can be refractory to allknown standard of care treatments. Typically, the subject has notpreviously been treated with the at least one treatment that isassociated with benefit for treatment of the cancer. Accordingly, themolecular profiling may reveal a new treatment option for theindividual.

Based on the results of the methods of the invention, the caregiver,e.g., a treating physician such as an oncologist, may determine atreatment regimen to the subject. In preferred embodiments, progressionfree survival (PFS), disease free survival (DFS), or lifespan isextended by administration of the at least one treatment that isassociated with benefit for treatment of the cancer to the individual.

The methods of the invention can be used to determine a molecularprofile for any desired cancer. The cancer may comprise withoutlimitation an acute lymphoblastic leukemia; acute myeloid leukemia;adrenocortical carcinoma; AIDS-related cancer; AIDS-related lymphoma;anal cancer; appendix cancer; astrocytomas; atypical teratoid/rhabdoidtumor; basal cell carcinoma; bladder cancer; brain stem glioma; braintumor, brain stem glioma, central nervous system atypicalteratoid/rhabdoid tumor, central nervous system embryonal tumors,astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma,medulloblastoma, medulloepithelioma, pineal parenchymal tumors ofintermediate differentiation, supratentorial primitive neuroectodermaltumors and pineoblastoma; breast cancer; bronchial tumors; Burkittlymphoma; cancer of unknown primary site (CUP); carcinoid tumor;carcinoma of unknown primary site; central nervous system atypicalteratoid/rhabdoid tumor; central nervous system embryonal tumors;cervical cancer; childhood cancers; chordoma; chronic lymphocyticleukemia; chronic myelogenous leukemia; chronic myeloproliferativedisorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneousT-cell lymphoma; endocrine pancreas islet cell tumors; endometrialcancer; ependymoblastoma; ependymoma; esophageal cancer;esthesioneuroblastoma; Ewing sarcoma; extracranial germ cell tumor;extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladdercancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor;gastrointestinal stromal cell tumor; gastrointestinal stromal tumor(GIST); gestational trophoblastic tumor; glioma; hairy cell leukemia;head and neck cancer; heart cancer; Hodgkin lymphoma; hypopharyngealcancer; intraocular melanoma; islet cell tumors; Kaposi sarcoma; kidneycancer; Langerhans cell histiocytosis; laryngeal cancer; lip cancer;liver cancer; malignant fibrous histiocytoma bone cancer;medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma;Merkel cell skin carcinoma; mesothelioma; metastatic squamous neckcancer with occult primary; mouth cancer; multiple endocrine neoplasiasyndromes; multiple myeloma; multiple myeloma/plasma cell neoplasm;mycosis fungoides; myelodysplastic syndromes; myeloproliferativeneoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma;Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lungcancer; oral cancer; oral cavity cancer; oropharyngeal cancer;osteosarcoma; other brain and spinal cord tumors; ovarian cancer;ovarian epithelial cancer; ovarian germ cell tumor; ovarian lowmalignant potential tumor; pancreatic cancer; papillomatosis; paranasalsinus cancer; parathyroid cancer; pelvic cancer; penile cancer;pharyngeal cancer; pineal parenchymal tumors of intermediatedifferentiation; pineoblastoma; pituitary tumor; plasma cellneoplasm/multiple myeloma; pleuropulmonary blastoma; primary centralnervous system (CNS) lymphoma; primary hepatocellular liver cancer;prostate cancer; rectal cancer; renal cancer; renal cell (kidney)cancer; renal cell cancer; respiratory tract cancer; retinoblastoma;rhabdomyosarcoma; salivary gland cancer; Sézary syndrome; small celllung cancer; small intestine cancer; soft tissue sarcoma; squamous cellcarcinoma; squamous neck cancer; stomach (gastric) cancer;supratentorial primitive neuroectodermal tumors; T-cell lymphoma;testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroidcancer; transitional cell cancer; transitional cell cancer of the renalpelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer;uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer;Waldenstrom macroglobulinemia; or Wilm's tumor. In embodiments, thecancer comprises an acute myeloid leukemia (AML), breast carcinoma,cholangiocarcinoma, colorectal adenocarcinoma, extrahepatic bile ductadenocarcinoma, female genital tract malignancy, gastric adenocarcinoma,gastroesophageal adenocarcinoma, gastrointestinal stromal tumor (GIST),glioblastoma, head and neck squamous carcinoma, leukemia, liverhepatocellular carcinoma, low grade glioma, lung bronchioloalveolarcarcinoma (BAC), non-small cell lung cancer (NSCLC), lung small cellcancer (SCLC), lymphoma, male genital tract malignancy, malignantsolitary fibrous tumor of the pleura (MSFT), melanoma, multiple myeloma,neuroendocrine tumor, nodal diffuse large B-cell lymphoma, nonepithelial ovarian cancer (non-EOC), ovarian surface epithelialcarcinoma, pancreatic adenocarcinoma, pituitary carcinomas,oligodendroglioma, prostatic adenocarcinoma, retroperitoneal orperitoneal carcinoma, retroperitoneal or peritoneal sarcoma, smallintestinal malignancy, soft tissue tumor, thymic carcinoma, thyroidcarcinoma, or uveal melanoma.

In some embodiments, the cancer comprises a breast cancer, triplenegative breast cancer, metaplastic breast cancer (MpBC), head and necksquamous cell carcinoma (HNSCC), human papilloma virus (HPV)-positiveHNSCC, HPV-negative/TP53-mutated HNSCC, metastatic HNSCC, oropharyngealHNSCC, non-oropharyngeal HNSCC, a carcinoma, a sarcoma, a melanoma, aluminal A breast cancer, a luminal B breast cancer, HER2+ breast cancer,a high microsatellite instability (MSI-H) colorectal cancer, amicrosatellite stable colorectal cancer (MSS), non-small cell lungcancer (NSCLC), chordoma, or adrenal cortical carcinoma. The carcinomacan be a carcinoma of the breast, colon, lung, pancreas, prostate,Merkel cell, ovary, liver, endometrial, bladder, kidney or cancer ofunknown primary (CUP). The sarcoma can be a liposarcoma, chondrosarcoma,extraskeletal myxoid chondrosarcoma or uterine sarcoma. In someembodiments, the sarcoma comprises an alveolar soft part sarcoma (ASPS),angiosarcoma, breast angiosarcoma, chondrosarcoma, chordoma, clear cellsarcoma, desmoplastic small round cell tumor (DSRCT), epithelioidhemangioendothelioma (EHE), epithelioid sarcoma, endometrial stromalsarcoma (ESS), ewing sarcoma, fibromatosis, fibrosarcoma, giant celltumour, leiomyosarcoma (LMS), uterine LMS, liposarcoma, malignantfibrous histiocytoma (MFH/UPS), malignant peripheral nerve sheath tumor(MPNST), osteosarcoma, perivascular epithelioid cell tumor (PEComa),rhabdomyosarcoma, solitary fibrous tumor (SFT), synovial sarcoma,fibromyxoid sarcoma, fibrous hamartoma of infancy, hereditaryleiomyomatosis, angiomyolipoma, angiomyxoma, atypical spindle celllesion (with fibrohistiocytic differentiation), chondroblastoma,dendritic cell sarcoma, granular cell tumor, high grade myxoid sarcoma,high-grade myoepithelial carcinoma, hyalinizing fibroblastic sarcoma,inflammatory myofibroblastic sarcoma, interdigitating dendritic celltumor, intimal sarcoma, leiomyoma, lymphangitic sarcomatosis, malignantglomus tumor, malignant myoepithelioma, melanocytic neoplasm,mesenchymal neoplasm, mesenteric glomangioma, metastatic histocytoidneoplasm, myoepithelioma, myxoid sarcoma, myxoid stromal, neurilemmoma,phyllodes, rhabdoid, round cell, sarcoma not otherwise specified (NOS),sarcomatous mesothelioma, schwannoma, spindle and round cell sarcoma,spindle cell or spinocellular mesenchymal tumor.

In a related aspect, the invention provides a method of generating amolecular profiling report comprising preparing a report comprisingresults of the determining and identifying steps as described above. Insome embodiments, the report further comprises a list of the at leastone treatment that is associated with benefit for treatment of thecancer, a list of the at least one treatment that is associated withlack of benefit for treatment of the cancer, and/or a list of at leastone treatment that is associated with indeterminate benefit for treatingthe cancer. The report can further comprise identification of the atleast one treatment as standard of care or not for the cancer, e.g.,using guidelines such as NCCN for the cancer's lineage. In someembodiments, the report further comprises a list of clinical trials forwhich the subject is indicated and/or eligible based on the molecularprofile. FIGS. 29A-V present an illustrative report according to theinvention.

The report may comprise various listings and descriptions of themolecular profiling that was performed. In some embodiments, the reportfurther comprises a listing of at least one member of the plurality ofgenes or gene products assessed with description of the at least onemember. For example, such descriptions can be as provided in Table 6herein. In embodiments, the report comprises a listing of the laboratorytechniques used to assess the members of the plurality of genes or geneproducts. For example, the report can specify whether each member wasassessed by at least one of ISH, IHC, Next Generation sequencing, Sangersequencing, PCR, pyrosequencing and fragment analysis. The report canprovide an evidentiary level for each biomarker-drug association. Forexample, the report may comprises a list of evidence supporting theidentification of certain treatments as likely to benefit the patient,not benefit the patient, or having indeterminate benefit. See, e.g.,Table 10 and accompanying text herein.

The report can provide any desired combination of such information. Insome embodiments, the report further comprises: 1) a list of the genesand/or gene products in the molecular profile; 2) a description of themolecular profile of the genes and/or gene products as determined forthe subject; 3) a treatment associated with at least one of the genesand/or gene products in the molecular profile; and 4) and an indicationwhether each treatment is likely to benefit the patient, not benefit thepatient, or has indeterminate benefit. The description of the molecularprofile of the genes and/or gene products as determined for the subjectmay comprise the technique used to assess the gene and/or gene productsand the results of the assessment.

In preferred embodiments, the report is computer generated. For example,the can be a printed report or a computer file. The report can be madeaccessible via a web portal.

In still another related aspect, the invention provides use of a reagentin carrying out the methods of the invention, and/or use of a reagent inthe manufacture of a reagent or kit for carrying out the methods of theinvention. Relatedly, the invention provides a kit comprising a reagentfor carrying out the methods of the invention. The reagent can be anyuseful reagent for performing molecular profiling. For example, thereagent may comprise at least one of a reagent for extracting nucleicacid from a sample, a reagent for performing ISH, a reagent forperforming IHC, a reagent for performing PCR, a reagent for performingSanger sequencing, a reagent for performing next generation sequencing,a reagent for a DNA microarray, a reagent for performing pyrosequencing,a nucleic acid probe, a nucleic acid primer, an antibody, a reagent forperforming bisulfate treatment of nucleic acid, and a combinationthereof.

In yet another related aspect, the invention provides a report generatedby the methods of the invention. The report can be a report as describedabove. For example, the can be a printed report or a computer file. Thereport can be made accessible via a web portal. The invention alsoprovides a computer system for generating the report.

In an aspect, the invention provides a system for identifying at leastone treatment associated with a cancer in a subject, comprising: a) ahost server; b) a user interface for accessing the host server to accessand input data; c) a processor for processing the inputted data; d) amemory coupled to the processor for storing the processed data andinstructions for: accessing a molecular profile generated by the methodsof the invention and identifying, based on the molecular profile, atleast one of: i) at least one treatment that is associated with benefitfor treatment of the cancer; ii) at least one treatment that isassociated with lack of benefit for treatment of the cancer; and iii) atleast one treatment associated with a clinical trial; and e) a displayfor displaying the identified at least one of: i) at least one treatmentthat is associated with benefit for treatment of the cancer; ii) atleast one treatment that is associated with lack of benefit fortreatment of the cancer; and iii) at least one treatment associated witha clinical trial. The display may comprise a molecular profiling reportas described above.

In a related aspect, the invention provides a system for generating areport identifying a therapeutic agent for an individual with a cancer,comprising: a) at least one device configured to assay a plurality ofplurality of genes and/or gene products in a biological sample from theindividual to determine molecular profile test values for the pluralityof gene or gene products, wherein the plurality of genes and/or geneproducts is selected from any of those described above; b) at least onecomputer database comprising: i) a reference value for each of theplurality of gene or gene products; and ii) a listing of availabletherapeutic agents with efficacy known to be related to at least one ofthe plurality of gene or gene products; c) a computer-readable programcode comprising instructions to input the molecular profile test valuesand to compare the molecular profile test values with a correspondingreference value from the at least one computer database in (b)(i); d) acomputer-readable program code comprising instructions to access the atleast one computer database and to identify at least one therapeuticagent from the listing of available therapeutic agents in (b)(ii),wherein the comparison to the reference in (c) indicates a likelybenefit or lack benefit of the at least one therapeutic agent; and e) acomputer-readable program comprising instructions to generate a reportthat comprises a listing of the members of the plurality of genes and/orgene products for which the comparison to the reference value indicateda likely benefit or lack of benefit of the at least one therapeuticagent in (d) and the at least one therapeutic agent identified in (d).The at least one device may include at least one nucleic acid sequencingdevice. The at least one nucleic acid sequencing device can beconfigured to assess any number of desired characteristics, includingwithout limitation at least one of a mutation, a polymorphism, adeletion, an insertion, a substitution, a translocation, a fusion, abreak, a duplication, an amplification, a repeat, a copy numbervariation, a transcript variant or a splice variant. In someembodiments, the at least one nucleic acid sequencing device comprises aNext Generation Sequencing device. Such device may be able to detectmany if not all of these characteristics in a single assay.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the presentinvention will be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the invention are used, and the accompanying drawings ofwhich:

FIG. 1 illustrates a block diagram of an exemplary embodiment of asystem for determining individualized medical intervention for aparticular disease state that utilizes molecular profiling of apatient's biological specimen that is non disease specific.

FIG. 2 is a flowchart of an exemplary embodiment of a method fordetermining individualized medical intervention for a particular diseasestate that utilizes molecular profiling of a patient's biologicalspecimen that is non disease specific.

FIGS. 3A through 3D illustrate an exemplary patient profile report inaccordance with step 80 of FIG. 2.

FIG. 4 is a flowchart of an exemplary embodiment of a method foridentifying a drug therapy/agent capable of interacting with a target.

FIGS. 5-14 are flowcharts and diagrams illustrating various parts of aninformation-based personalized medicine drug discovery system and methodin accordance with the present invention.

FIGS. 15-25 are computer screen print outs associated with various partsof the information-based personalized medicine drug discovery system andmethod shown in FIGS. 5-14.

FIGS. 26A-D illustrate a molecular profiling service requisition using amolecular profiling approach as outlined in Tables 7-9 and 12-15, andaccompanying text herein.

FIGS. 27A-V illustrate an exemplary patient report based on molecularprofiling for a patient having a triple negative breast cancer.

FIG. 28 illustrates progression free survival (PFS) using therapyselected by molecular profiling (period B) with PFS for the most recenttherapy on which the patient has just progressed (period A). IfPFS(B)/PFS(A) ratio ≥1.3, then molecular profiling selected therapy wasdefined as having benefit for patient.

FIG. 29 is a schematic of methods for identifying treatments bymolecular profiling if a target is identified.

FIG. 30 illustrates the distribution of the patients in the study asperformed in Example 1.

FIG. 31 is graph depicting the results of the study with patients havingPFS ratio ≥1.3 was 18/66 (27%).

FIG. 32 is a waterfall plot of all the patients for maximum % change ofsummed diameters of target lesions with respect to baseline diameter.

FIG. 33 illustrates the relationship between what clinician selected aswhat she/he would use to treat the patient before knowing what themolecular profiling results suggested. There were no matches for the 18patients with PFS ratio ≥1.3.

FIG. 34 is a schematic of the overall survival for the 18 patients withPFS ratio ≥1.3 versus all 66 patients.

FIG. 35 illustrates a molecular profiling system that performs analysisof a cancer sample using a variety of components that measure expressionlevels, chromosomal aberrations and mutations. The molecular “blueprint”of the cancer is used to generate a prioritized ranking of druggabletargets and/or drug associated targets in tumor and their associatedtherapies.

FIG. 36 shows an example output of microarray profiling results andcalls made using a cutoff value.

FIGS. 37A-F illustrate results of molecular profiling of a cohort of 126Triple Negative (TN) Metaplastic Breast Cancers.

FIG. 38 illustrates results of molecular profiling of PD1 and PDL1 inHPV+ and HPV−/TP53 mutated head and neck squamous cell carcinomas.

FIGS. 39A-D illustrates a case of endometrial adenocarcinoma (FIG. 39A,hematoxylin and eosin stained section) exhibiting microsatelliteinstability caused by the loss of MLH-1 protein [note retained MLH-1protein expression in the nuclei of the tumor infiltrating lymphocytes](FIG. 39B, immunohistochemical stain); PD-1+ Tumor-infiltratinglymphocytes (FIG. 39C, immunohistochemical stain); aberrant expressionof PD-L1 in the tumor cells' basolateral membranes (FIG. 39D,immunohistochemical stain).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and systems for identifyingtherapeutic agents for use in treatments on an individualized basis byusing molecular profiling. The molecular profiling approach provides amethod for selecting a candidate treatment for an individual that couldfavorably change the clinical course for the individual with a conditionor disease, such as cancer. The molecular profiling approach providesclinical benefit for individuals, such as identifying drug target(s)that provide a longer progression free survival (PFS), longer diseasefree survival (DFS), longer overall survival (OS) or extended lifespan.Methods and systems of the invention are directed to molecular profilingof cancer on an individual basis that can provide alternatives fortreatment that may be convention or alternative to conventionaltreatment regimens. For example, alternative treatment regimes can beselected through molecular profiling methods of the invention where, adisease is refractory to current therapies, e.g., after a cancer hasdeveloped resistance to a standard-of-care treatment. Illustrativeschemes for using molecular profiling to identify a treatment regime areshown in FIGS. 2, 49A-B and 50, each of which is described in furtherdetail herein. Thus, molecular profiling provides a personalizedapproach to selecting candidate treatments that are likely to benefit acancer. In embodiments, the molecular profiling method is used toidentify therapies for patients with poor prognosis, such as those withmetastatic disease or those whose cancer has progressed on standardfront line therapies, or whose cancer has progressed on multiplechemotherapeutic or hormonal regimens.

Personalized medicine based on pharmacogenetic insights, such as thoseprovided by molecular profiling according to the invention, isincreasingly taken for granted by some practitioners and the lay press,but forms the basis of hope for improved cancer therapy. However,molecular profiling as taught herein represents a fundamental departurefrom the traditional approach to oncologic therapy where for the mostpart, patients are grouped together and treated with approaches that arebased on findings from light microscopy and disease stage.Traditionally, differential response to a particular therapeuticstrategy has only been determined after the treatment was given, i.e. aposteriori. The “standard” approach to disease treatment relies on whatis generally true about a given cancer diagnosis and treatment responsehas been vetted by randomized phase III clinical trials and forms the“standard of care” in medical practice. The results of these trials havebeen codified in consensus statements by guidelines organizations suchas the National Comprehensive Cancer Network and The American Society ofClinical Oncology. The NCCN Compendium™ contains authoritative,scientifically derived information designed to support decision-makingabout the appropriate use of drugs and biologics in patients withcancer. The NCCN Compendium™ is recognized by the Centers for Medicareand Medicaid Services (CMS) and United Healthcare as an authoritativereference for oncology coverage policy. On-compendium treatments arethose recommended by such guides. The biostatistical methods used tovalidate the results of clinical trials rely on minimizing differencesbetween patients, and are based on declaring the likelihood of errorthat one approach is better than another for a patient group definedonly by light microscopy and stage, not by individual differences intumors. The molecular profiling methods of the invention exploit suchindividual differences. The methods can provide candidate treatmentsthat can be then selected by a physician for treating a patient. In astudy of such an approach presented in Example 1 herein, the resultswere profound: in 66 consecutive patients, the treating oncologist nevermanaged to identify the molecular target selected by the test, and 27%of patients whose treatment was guided by molecular profiling managed aremission 1.3× longer than their previous best response. At present,such results are virtually unheard of result in the salvage therapysetting.

Molecular profiling can be used to provide a comprehensive view of thebiological state of a sample. In an embodiment, molecular profiling isused for whole tumor profiling. Accordingly, a number of molecularapproaches are used to assess the state of a tumor. The whole tumorprofiling can be used for selecting a candidate treatment for a tumor.Molecular profiling can be used to select candidate therapeutics on anysample for any stage of a disease. In embodiment, the methods of theinvention are used to profile a newly diagnosed cancer. The candidatetreatments indicated by the molecular profiling can be used to select atherapy for treating the newly diagnosed cancer. In other embodiments,the methods of the invention are used to profile a cancer that hasalready been treated, e.g., with one or more standard-of-care therapy.In embodiments, the cancer is refractory to the prior treatment/s. Forexample, the cancer may be refractory to the standard of care treatmentsfor the cancer. The cancer can be a metastatic cancer or other recurrentcancer. The treatments can be on-compendium or off-compendiumtreatments.

Molecular profiling can be performed by any known means for detecting amolecule in a biological sample. Molecular profiling comprises methodsthat include but are not limited to, nucleic acid sequencing, such as aDNA sequencing or mRNA sequencing; immunohistochemistry (IHC); in situhybridization (ISH); fluorescent in situ hybridization (FISH);chromogenic in situ hybridization (CISH); PCR amplification (e.g., qPCRor RT-PCR); various types of microarray (mRNA expression arrays, lowdensity arrays, protein arrays, etc); various types of sequencing(Sanger, pyrosequencing, etc); comparative genomic hybridization (CGH);NextGen sequencing; Northern blot; Southern blot; immunoassay; and anyother appropriate technique to assay the presence or quantity of abiological molecule of interest. In various embodiments of theinvention, any one or more of these methods can be used concurrently orsubsequent to each other for assessing target genes disclosed herein.

Molecular profiling of individual samples is used to select one or morecandidate treatments for a disorder in a subject, e.g., by identifyingtargets for drugs that may be effective for a given cancer. For example,the candidate treatment can be a treatment known to have an effect oncells that differentially express genes as identified by molecularprofiling techniques, an experimental drug, a government or regulatoryapproved drug or any combination of such drugs, which may have beenstudied and approved for a particular indication that is the same as ordifferent from the indication of the subject from whom a biologicalsample is obtain and molecularly profiled.

When multiple biomarker targets are revealed by assessing target genesby molecular profiling, one or more decision rules can be put in placeto prioritize the selection of certain therapeutic agent for treatmentof an individual on a personalized basis. Rules of the invention aideprioritizing treatment, e.g., direct results of molecular profiling,anticipated efficacy of therapeutic agent, prior history with the sameor other treatments, expected side effects, availability of therapeuticagent, cost of therapeutic agent, drug-drug interactions, and otherfactors considered by a treating physician. Based on the recommended andprioritized therapeutic agent targets, a physician can decide on thecourse of treatment for a particular individual. Accordingly, molecularprofiling methods and systems of the invention can select candidatetreatments based on individual characteristics of diseased cells, e.g.,tumor cells, and other personalized factors in a subject in need oftreatment, as opposed to relying on a traditional one-size fits allapproach that is conventionally used to treat individuals suffering froma disease, especially cancer. In some cases, the recommended treatmentsare those not typically used to treat the disease or disorder inflictingthe subject. In some cases, the recommended treatments are used afterstandard-of-care therapies are no longer providing adequate efficacy.

The treating physician can use the results of the molecular profilingmethods to optimize a treatment regimen for a patient. The candidatetreatment identified by the methods of the invention can be used totreat a patient; however, such treatment is not required of the methods.Indeed, the analysis of molecular profiling results and identificationof candidate treatments based on those results can be automated and doesnot require physician involvement.

Biological Entities

Nucleic acids include deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, orcomplements thereof. Nucleic acids can contain known nucleotide analogsor modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, which have similarbinding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Nucleic acidsequence can encompass conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell Probes 8:91-98 (1994)). The termnucleic acid can be used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

A particular nucleic acid sequence may implicitly encompass theparticular sequence and “splice variants” and nucleic acid sequencesencoding truncated forms. Similarly, a particular protein encoded by anucleic acid can encompass any protein encoded by a splice variant ortruncated form of that nucleic acid. “Splice variants,” as the namesuggests, are products of alternative splicing of a gene. Aftertranscription, an initial nucleic acid transcript may be spliced suchthat different (alternate) nucleic acid splice products encode differentpolypeptides. Mechanisms for the production of splice variants vary, butinclude alternate splicing of exons. Alternate polypeptides derived fromthe same nucleic acid by read-through transcription are also encompassedby this definition. Any products of a splicing reaction, includingrecombinant forms of the splice products, are included in thisdefinition. Nucleic acids can be truncated at the 5′ end or at the 3′end. Polypeptides can be truncated at the N-terminal end or theC-terminal end. Truncated versions of nucleic acid or polypeptidesequences can be naturally occurring or created using recombinanttechniques.

The terms “genetic variant” and “nucleotide variant” are used hereininterchangeably to refer to changes or alterations to the referencehuman gene or cDNA sequence at a particular locus, including, but notlimited to, nucleotide base deletions, insertions, inversions, andsubstitutions in the coding and non-coding regions. Deletions may be ofa single nucleotide base, a portion or a region of the nucleotidesequence of the gene, or of the entire gene sequence. Insertions may beof one or more nucleotide bases. The genetic variant or nucleotidevariant may occur in transcriptional regulatory regions, untranslatedregions of mRNA, exons, introns, exon/intron junctions, etc. The geneticvariant or nucleotide variant can potentially result in stop codons,frame shifts, deletions of amino acids, altered gene transcript spliceforms or altered amino acid sequence.

An allele or gene allele comprises generally a naturally occurring genehaving a reference sequence or a gene containing a specific nucleotidevariant.

A haplotype refers to a combination of genetic (nucleotide) variants ina region of an mRNA or a genomic DNA on a chromosome found in anindividual. Thus, a haplotype includes a number of genetically linkedpolymorphic variants which are typically inherited together as a unit.

As used herein, the term “amino acid variant” is used to refer to anamino acid change to a reference human protein sequence resulting fromgenetic variants or nucleotide variants to the reference human geneencoding the reference protein. The term “amino acid variant” isintended to encompass not only single amino acid substitutions, but alsoamino acid deletions, insertions, and other significant changes of aminoacid sequence in the reference protein.

The term “genotype” as used herein means the nucleotide characters at aparticular nucleotide variant marker (or locus) in either one allele orboth alleles of a gene (or a particular chromosome region). With respectto a particular nucleotide position of a gene of interest, thenucleotide(s) at that locus or equivalent thereof in one or both allelesform the genotype of the gene at that locus. A genotype can behomozygous or heterozygous. Accordingly, “genotyping” means determiningthe genotype, that is, the nucleotide(s) at a particular gene locus.Genotyping can also be done by determining the amino acid variant at aparticular position of a protein which can be used to deduce thecorresponding nucleotide variant(s).

The term “locus” refers to a specific position or site in a genesequence or protein. Thus, there may be one or more contiguousnucleotides in a particular gene locus, or one or more amino acids at aparticular locus in a polypeptide. Moreover, a locus may refer to aparticular position in a gene where one or more nucleotides have beendeleted, inserted, or inverted.

Unless specified otherwise or understood by one of skill in art, theterms “polypeptide,” “protein,” and “peptide” are used interchangeablyherein to refer to an amino acid chain in which the amino acid residuesare linked by covalent peptide bonds. The amino acid chain can be of anylength of at least two amino acids, including full-length proteins.Unless otherwise specified, polypeptide, protein, and peptide alsoencompass various modified forms thereof, including but not limited toglycosylated forms, phosphorylated forms, etc. A polypeptide, protein orpeptide can also be referred to as a gene product.

Lists of gene and gene products that can be assayed by molecularprofiling techniques are presented herein. Lists of genes may bepresented in the context of molecular profiling techniques that detect agene product (e.g., an mRNA or protein). One of skill will understandthat this implies detection of the gene product of the listed genes.Similarly, lists of gene products may be presented in the context ofmolecular profiling techniques that detect a gene sequence or copynumber. One of skill will understand that this implies detection of thegene corresponding to the gene products, including as an example DNAencoding the gene products. As will be appreciated by those skilled inthe art, a “biomarker” or “marker” comprises a gene and/or gene productdepending on the context.

The terms “label” and “detectable label” can refer to any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical, chemical or similar methods. Such labels includebiotin for staining with labeled streptavidin conjugate, magnetic beads(e.g., DYNABEADS™) fluorescent dyes (e.g., fluorescein, Texas red,rhodamine, green fluorescent protein, and the like), radiolabels (e.g.,³H, ¹²⁵I ³⁵S, ¹⁴C or ³²P), enzymes (e.g., horse radish peroxidase,alkaline phosphatase and others commonly used in an ELISA), andcalorimetric labels such as colloidal gold or colored glass or plastic(e.g., polystyrene, polypropylene, latex, etc) beads. Patents teachingthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Means ofdetecting such labels are well known to those of skill in the art. Thus,for example, radiolabels may be detected using photographic film orscintillation counters, fluorescent markers may be detected using aphotodetector to detect emitted light. Enzymatic labels are typicallydetected by providing the enzyme with a substrate and detecting thereaction product produced by the action of the enzyme on the substrate,and calorimetric labels are detected by simply visualizing the coloredlabel. Labels can include, e.g., ligands that bind to labeledantibodies, fluorophores, chemiluminescent agents, enzymes, andantibodies which can serve as specific binding pair members for alabeled ligand. An introduction to labels, labeling procedures anddetection of labels is found in Polak and Van Noorden Introduction toImmunocytochemistry, 2nd ed., Springer Verlag, NY (1997); and inHaugland Handbook of Fluorescent Probes and Research Chemicals, acombined handbook and catalogue Published by Molecular Probes, Inc.(1996).

Detectable labels include, but are not limited to, nucleotides (labeledor unlabelled), compomers, sugars, peptides, proteins, antibodies,chemical compounds, conducting polymers, binding moieties such asbiotin, mass tags, calorimetric agents, light emitting agents,chemiluminescent agents, light scattering agents, fluorescent tags,radioactive tags, charge tags (electrical or magnetic charge), volatiletags and hydrophobic tags, biomolecules (e.g., members of a binding pairantibody/antigen, antibody/antibody, antibody/antibody fragment,antibody/antibody receptor, antibody/protein A or protein G,hapten/anti-hapten, biotin/avidin, biotin/streptavidin, folicacid/folate binding protein, vitamin B12/intrinsic factor, chemicalreactive group/complementary chemical reactive group (e.g.,sulfhydryl/maleimide, sulfhydryl/haloacetyl derivative,amine/isotriocyanate, amine/succinimidyl ester, and amine/sulfonylhalides) and the like.

The term “antibody” as used herein encompasses naturally occurringantibodies as well as non-naturally occurring antibodies, including, forexample, single chain antibodies, chimeric, bifunctional and humanizedantibodies, as well as antigen-binding fragments thereof, (e.g., Fab′,F(ab′)₂, Fab, Fv and rIgG). See also, Pierce Catalog and Handbook,1994-1995 (Pierce Chemical Co., Rockford, Ill.). See also, e.g., Kuby,J., Immunology, 3.sup.rd Ed., W. H. Freeman & Co., New York (1998). Suchnon-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly or can be obtained, forexample, by screening combinatorial libraries consisting of variableheavy chains and variable light chains as described by Huse et al.,Science 246:1275-1281 (1989), which is incorporated herein by reference.These and other methods of making, for example, chimeric, humanized,CDR-grafted, single chain, and bifunctional antibodies are well known tothose skilled in the art. See, e.g., Winter and Harris, Immunol. Today14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow andLane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications,New York, 1988; Hilyard et al., Protein Engineering: A practicalapproach (IRL Press 1992); Borrebaeck, Antibody Engineering, 2d ed.(Oxford University Press 1995); each of which is incorporated herein byreference.

Unless otherwise specified, antibodies can include both polyclonal andmonoclonal antibodies. Antibodies also include genetically engineeredforms such as chimeric antibodies (e.g., humanized murine antibodies)and heteroconjugate antibodies (e.g., bispecific antibodies). The termalso refers to recombinant single chain Fv fragments (scFv). The termantibody also includes bivalent or bispecific molecules, diabodies,triabodies, and tetrabodies. Bivalent and bispecific molecules aredescribed in, e.g., Kostelny et al. (1992) J Immunol 148:1547, Pack andPluckthun (1992) Biochemistry 31:1579, Holliger et al. (1993) Proc NatlAcad Sci USA. 90:6444, Gruber et al. (1994) J Immunol: 5368, Zhu et al.(1997) Protein Sci 6:781, Hu et al. (1997) Cancer Res. 56:3055, Adams etal. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) ProteinEng. 8:301.

Typically, an antibody has a heavy and light chain. Each heavy and lightchain contains a constant region and a variable region, (the regions arealso known as “domains”). Light and heavy chain variable regions containfour framework regions interrupted by three hyper-variable regions, alsocalled complementarity-determining regions (CDRs). The extent of theframework regions and CDRs have been defined. The sequences of theframework regions of different light or heavy chains are relativelyconserved within a species. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs in three dimensionalspaces. The CDRs are primarily responsible for binding to an epitope ofan antigen. The CDRs of each chain are typically referred to as CDR1,CDR2, and CDR3, numbered sequentially starting from the N-terminus, andare also typically identified by the chain in which the particular CDRis located. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. References to V_(H) refer to the variable regionof an immunoglobulin heavy chain of an antibody, including the heavychain of an Fv, scFv, or Fab. References to V_(L) refer to the variableregion of an immunoglobulin light chain, including the light chain of anFv, scFv, dsFv or Fab.

The phrase “single chain Fv” or “scFv” refers to an antibody in whichthe variable domains of the heavy chain and of the light chain of atraditional two chain antibody have been joined to form one chain.Typically, a linker peptide is inserted between the two chains to allowfor proper folding and creation of an active binding site. A “chimericantibody” is an immunoglobulin molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

A “humanized antibody” is an immunoglobulin molecule that containsminimal sequence derived from non-human immunoglobulin. Humanizedantibodies include human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, a humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the framework(FR) regions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992)). Humanization can be essentially performed followingthe method of Winter and co-workers (Jones et al., Nature 321:522-525(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody.Accordingly, such humanized antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species.

The terms “epitope” and “antigenic determinant” refer to a site on anantigen to which an antibody binds. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

The terms “primer”, “probe,” and “oligonucleotide” are used hereininterchangeably to refer to a relatively short nucleic acid fragment orsequence. They can comprise DNA, RNA, or a hybrid thereof, or chemicallymodified analog or derivatives thereof. Typically, they aresingle-stranded. However, they can also be double-stranded having twocomplementing strands which can be separated by denaturation. Normally,primers, probes and oligonucleotides have a length of from about 8nucleotides to about 200 nucleotides, preferably from about 12nucleotides to about 100 nucleotides, and more preferably about 18 toabout 50 nucleotides. They can be labeled with detectable markers ormodified using conventional manners for various molecular biologicalapplications.

The term “isolated” when used in reference to nucleic acids (e.g.,genomic DNAs, cDNAs, mRNAs, or fragments thereof) is intended to meanthat a nucleic acid molecule is present in a form that is substantiallyseparated from other naturally occurring nucleic acids that are normallyassociated with the molecule. Because a naturally existing chromosome(or a viral equivalent thereof) includes a long nucleic acid sequence,an isolated nucleic acid can be a nucleic acid molecule having only aportion of the nucleic acid sequence in the chromosome but not one ormore other portions present on the same chromosome. More specifically,an isolated nucleic acid can include naturally occurring nucleic acidsequences that flank the nucleic acid in the naturally existingchromosome (or a viral equivalent thereof). An isolated nucleic acid canbe substantially separated from other naturally occurring nucleic acidsthat are on a different chromosome of the same organism. An isolatednucleic acid can also be a composition in which the specified nucleicacid molecule is significantly enriched so as to constitute at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of thetotal nucleic acids in the composition.

An isolated nucleic acid can be a hybrid nucleic acid having thespecified nucleic acid molecule covalently linked to one or more nucleicacid molecules that are not the nucleic acids naturally flanking thespecified nucleic acid. For example, an isolated nucleic acid can be ina vector. In addition, the specified nucleic acid may have a nucleotidesequence that is identical to a naturally occurring nucleic acid or amodified form or mutein thereof having one or more mutations such asnucleotide substitution, deletion/insertion, inversion, and the like.

An isolated nucleic acid can be prepared from a recombinant host cell(in which the nucleic acids have been recombinantly amplified and/orexpressed), or can be a chemically synthesized nucleic acid having anaturally occurring nucleotide sequence or an artificially modified formthereof.

The term “isolated polypeptide” as used herein is defined as apolypeptide molecule that is present in a form other than that found innature. Thus, an isolated polypeptide can be a non-naturally occurringpolypeptide. For example, an isolated polypeptide can be a “hybridpolypeptide.” An isolated polypeptide can also be a polypeptide derivedfrom a naturally occurring polypeptide by additions or deletions orsubstitutions of amino acids. An isolated polypeptide can also be a“purified polypeptide” which is used herein to mean a composition orpreparation in which the specified polypeptide molecule is significantlyenriched so as to constitute at least 10% of the total protein contentin the composition. A “purified polypeptide” can be obtained fromnatural or recombinant host cells by standard purification techniques,or by chemically synthesis, as will be apparent to skilled artisans.

The terms “hybrid protein,” “hybrid polypeptide,” “hybrid peptide,”“fusion protein,” “fusion polypeptide,” and “fusion peptide” are usedherein interchangeably to mean a non-naturally occurring polypeptide orisolated polypeptide having a specified polypeptide molecule covalentlylinked to one or more other polypeptide molecules that do not link tothe specified polypeptide in nature. Thus, a “hybrid protein” may be twonaturally occurring proteins or fragments thereof linked together by acovalent linkage. A “hybrid protein” may also be a protein formed bycovalently linking two artificial polypeptides together. Typically butnot necessarily, the two or more polypeptide molecules are linked or“fused” together by a peptide bond forming a single non-branchedpolypeptide chain.

The term “high stringency hybridization conditions,” when used inconnection with nucleic acid hybridization, includes hybridizationconducted overnight at 42° C. in a solution containing 50% formamide,5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate, pH7.6, 5×Denhardt's solution, 10% dextran sulfate, and 20 microgram/mldenatured and sheared salmon sperm DNA, with hybridization filterswashed in 0.1×SSC at about 65° C. The term “moderate stringenthybridization conditions,” when used in connection with nucleic acidhybridization, includes hybridization conducted overnight at 37° C. in asolution containing 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodiumcitrate), 50 mM sodium phosphate, pH 7.6, 5×Denhardt's solution, 10%dextran sulfate, and 20 microgram/ml denatured and sheared salmon spermDNA, with hybridization filters washed in 1×SSC at about 50° C. It isnoted that many other hybridization methods, solutions and temperaturescan be used to achieve comparable stringent hybridization conditions aswill be apparent to skilled artisans.

For the purpose of comparing two different nucleic acid or polypeptidesequences, one sequence (test sequence) may be described to be aspecific percentage identical to another sequence (comparison sequence).The percentage identity can be determined by the algorithm of Karlin andAltschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993), which isincorporated into various BLAST programs. The percentage identity can bedetermined by the “BLAST 2 Sequences” tool, which is available at theNational Center for Biotechnology Information (NCBI) website. SeeTatusova and Madden, FEMS Microbiol. Lett., 174(2):247-250 (1999). Forpairwise DNA-DNA comparison, the BLASTN program is used with defaultparameters (e.g., Match: 1; Mismatch: −2; Open gap: 5 penalties;extension gap: 2 penalties; gap x_dropoff: 50; expect: 10; and wordsize: 11, with filter). For pairwise protein-protein sequencecomparison, the BLASTP program can be employed using default parameters(e.g., Matrix: BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 15;expect: 10.0; and wordsize: 3, with filter). Percent identity of twosequences is calculated by aligning a test sequence with a comparisonsequence using BLAST, determining the number of amino acids ornucleotides in the aligned test sequence that are identical to aminoacids or nucleotides in the same position of the comparison sequence,and dividing the number of identical amino acids or nucleotides by thenumber of amino acids or nucleotides in the comparison sequence. WhenBLAST is used to compare two sequences, it aligns the sequences andyields the percent identity over defined, aligned regions. If the twosequences are aligned across their entire length, the percent identityyielded by the BLAST is the percent identity of the two sequences. IfBLAST does not align the two sequences over their entire length, thenthe number of identical amino acids or nucleotides in the unalignedregions of the test sequence and comparison sequence is considered to bezero and the percent identity is calculated by adding the number ofidentical amino acids or nucleotides in the aligned regions and dividingthat number by the length of the comparison sequence. Various versionsof the BLAST programs can be used to compare sequences, e.g., BLAST2.1.2 or BLAST+2.2.22.

A subject or individual can be any animal which may benefit from themethods of the invention, including, e.g., humans and non-human mammals,such as primates, rodents, horses, dogs and cats. Subjects includewithout limitation a eukaryotic organisms, most preferably a mammal suchas a primate, e.g., chimpanzee or human, cow; dog; cat; a rodent, e.g.,guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish. Subjectsspecifically intended for treatment using the methods described hereininclude humans. A subject may be referred to as an individual or apatient.

Treatment of a disease or individual according to the invention is anapproach for obtaining beneficial or desired medical results, includingclinical results, but not necessarily a cure. For purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, alleviation or amelioration of one or more symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, preventing spread of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. Treatment also includes prolonging survival as compared toexpected survival if not receiving treatment or if receiving a differenttreatment. A treatment can include administration of a therapeuticagent, which can be an agent that exerts a cytotoxic, cytostatic, orimmunomodulatory effect on diseased cells, e.g., cancer cells, or othercells that may promote a diseased state, e.g., activated immune cells.Therapeutic agents selected by the methods of the invention are notlimited. Any therapeutic agent can be selected where a link can be madebetween molecular profiling and potential efficacy of the agent.Therapeutic agents include without limitation drugs, pharmaceuticals,small molecules, protein therapies, antibody therapies, viral therapies,gene therapies, and the like. Cancer treatments or therapies includeapoptosis-mediated and non-apoptosis mediated cancer therapiesincluding, without limitation, chemotherapy, hormonal therapy,radiotherapy, immunotherapy, and combinations thereof. Chemotherapeuticagents comprise therapeutic agents and combinations of therapeuticagents that treat, cancer cells, e.g., by killing those cells. Examplesof different types of chemotherapeutic drugs include without limitationalkylating agents (e.g., nitrogen mustard derivatives, ethylenimines,alkylsulfonates, hydrazines and triazines, nitrosureas, and metalsalts), plant alkaloids (e.g., vinca alkaloids, taxanes,podophyllotoxins, and camptothecan analogs), antitumor antibiotics(e.g., anthracyclines, chromomycins, and the like), antimetabolites(e.g., folic acid antagonists, pyrimidine antagonists, purineantagonists, and adenosine deaminase inhibitors), topoisomerase Iinhibitors, topoisomerase II inhibitors, and miscellaneousantineoplastics (e.g., ribonucleotide reductase inhibitors,adrenocortical steroid inhibitors, enzymes, antimicrotubule agents, andretinoids).

A biomarker refers generally to a molecule, including without limitationa gene or product thereof, nucleic acids (e.g., DNA, RNA),protein/peptide/polypeptide, carbohydrate structure, lipid, glycolipid,characteristics of which can be detected in a tissue or cell to provideinformation that is predictive, diagnostic, prognostic and/ortheranostic for sensitivity or resistance to candidate treatment.

Biological Samples

A sample as used herein includes any relevant biological sample that canbe used for molecular profiling, e.g., sections of tissues such asbiopsy or tissue removed during surgical or other procedures, bodilyfluids, autopsy samples, and frozen sections taken for histologicalpurposes. Such samples include blood and blood fractions or products(e.g., serum, buffy coat, plasma, platelets, red blood cells, and thelike), sputum, malignant effusion, cheek cells tissue, cultured cells(e.g., primary cultures, explants, and transformed cells), stool, urine,other biological or bodily fluids (e.g., prostatic fluid, gastric fluid,intestinal fluid, renal fluid, lung fluid, cerebrospinal fluid, and thelike), etc. The sample can comprise biological material that is a freshfrozen & formalin fixed paraffin embedded (FFPE) block, formalin-fixedparaffin embedded, or is within an RNA preservative+formalin fixative.More than one sample of more than one type can be used for each patient.In a preferred embodiment, the sample comprises a fixed tumor sample.

The sample used in the methods described herein can be a formalin fixedparaffin embedded (FFPE) sample. The FFPE sample can be one or more offixed tissue, unstained slides, bone marrow core or clot, core needlebiopsy, malignant fluids and fine needle aspirate (FNA). In anembodiment, the fixed tissue comprises a tumor containing formalin fixedparaffin embedded (FFPE) block from a surgery or biopsy. In anotherembodiment, the unstained slides comprise unstained, charged, unbakedslides from a paraffin block. In another embodiment, bone marrow core orclot comprises a decalcified core. A formalin fixed core and/or clot canbe paraffin-embedded. In still another embodiment, the core needlebiopsy comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, e.g., 3-4,paraffin embedded biopsy samples. An 18 gauge needle biopsy can be used.The malignant fluid can comprise a sufficient volume of freshpleural/ascitic fluid to produce a 5×5×2 mm cell pellet. The fluid canbe formalin fixed in a paraffin block. In an embodiment, the core needlebiopsy comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, e.g., 4-6,paraffin embedded aspirates.

A sample may be processed according to techniques understood by those inthe art. A sample can be without limitation fresh, frozen or fixed cellsor tissue. In some embodiments, a sample comprises formalin-fixedparaffin-embedded (FFPE) tissue, fresh tissue or fresh frozen (FF)tissue. A sample can comprise cultured cells, including primary orimmortalized cell lines derived from a subject sample. A sample can alsorefer to an extract from a sample from a subject. For example, a samplecan comprise DNA, RNA or protein extracted from a tissue or a bodilyfluid. Many techniques and commercial kits are available for suchpurposes. The fresh sample from the individual can be treated with anagent to preserve RNA prior to further processing, e.g., cell lysis andextraction. Samples can include frozen samples collected for otherpurposes. Samples can be associated with relevant information such asage, gender, and clinical symptoms present in the subject; source of thesample; and methods of collection and storage of the sample. A sample istypically obtained from a subject.

A biopsy comprises the process of removing a tissue sample fordiagnostic or prognostic evaluation, and to the tissue specimen itself.Any biopsy technique known in the art can be applied to the molecularprofiling methods of the present invention. The biopsy technique appliedcan depend on the tissue type to be evaluated (e.g., colon, prostate,kidney, bladder, lymph node, liver, bone marrow, blood cell, lung,breast, etc.), the size and type of the tumor (e.g., solid or suspended,blood or ascites), among other factors. Representative biopsy techniquesinclude, but are not limited to, excisional biopsy, incisional biopsy,needle biopsy, surgical biopsy, and bone marrow biopsy. An “excisionalbiopsy” refers to the removal of an entire tumor mass with a smallmargin of normal tissue surrounding it. An “incisional biopsy” refers tothe removal of a wedge of tissue that includes a cross-sectionaldiameter of the tumor. Molecular profiling can use a “core-needlebiopsy” of the tumor mass, or a “fine-needle aspiration biopsy” whichgenerally obtains a suspension of cells from within the tumor mass.Biopsy techniques are discussed, for example, in Harrison's Principlesof Internal Medicine, Kasper, et al., eds., 16th ed., 2005, Chapter 70,and throughout Part V.

Standard molecular biology techniques known in the art and notspecifically described are generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York (1989), and as in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and as inPerbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, NewYork (1988), and as in Watson et al., Recombinant DNA, ScientificAmerican Books, New York and in Birren et al (eds) Genome Analysis: ALaboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press,New York (1998) and methodology as set forth in U.S. Pat. Nos.4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 andincorporated herein by reference. Polymerase chain reaction (PCR) can becarried out generally as in PCR Protocols: A Guide to Methods andApplications, Academic Press, San Diego, Calif. (1990).

Vesicles

The sample can comprise vesicles. Methods of the invention can includeassessing one or more vesicles, including assessing vesicle populations.A vesicle, as used herein, is a membrane vesicle that is shed fromcells. Vesicles or membrane vesicles include without limitation:circulating microvesicles (cMVs), microvesicle, exosome, nanovesicle,dexosome, bleb, blebby, prostasome, microparticle, intralumenal vesicle,membrane fragment, intralumenal endosomal vesicle, endosomal-likevesicle, exocytosis vehicle, endosome vesicle, endosomal vesicle,apoptotic body, multivesicular body, secretory vesicle, phospholipidvesicle, liposomal vesicle, argosome, texasome, secresome, tolerosome,melanosome, oncosome, or exocytosed vehicle. Furthermore, althoughvesicles may be produced by different cellular processes, the methods ofthe invention are not limited to or reliant on any one mechanism,insofar as such vesicles are present in a biological sample and arecapable of being characterized by the methods disclosed herein. Unlessotherwise specified, methods that make use of a species of vesicle canbe applied to other types of vesicles. Vesicles comprise sphericalstructures with a lipid bilayer similar to cell membranes whichsurrounds an inner compartment which can contain soluble components,sometimes referred to as the payload. In some embodiments, the methodsof the invention make use of exosomes, which are small secreted vesiclesof about 40-100 nm in diameter. For a review of membrane vesicles,including types and characterizations, see Thery et al., Nat RevImmunol. 2009 August; 9(8):581-93. Some properties of different types ofvesicles include those in Table 1:

TABLE 1 Vesicle Properties Membrane Exosome-like Apoptotic FeatureExosomes Microvesicles Ectosomes particles vesicles vesicles Size 50-100nm 100-1,000 nm 50-200 nm 50-80 nm 20-50 nm 50-500 nm Density in1.13-1.19 g/ml 1.04-1.07 g/ml 1.1 g/ml 1.16-1.28 g/ml sucrose EM Cupshape Irregular Bilamellar Round Irregular Heterogeneous appearanceshape, round shape electron structures dense Sedimentation 100,000 g10,000 g 160.000-200.000 g 100.000-200,000 g 175,000 g 1,200 g, 10,000g, 100,000 g Lipid Enriched in Expose PPS Enriched in No lipidcomposition cholesterol, cholesterol rafts sphingomyelin and andceramide; diacylglycerol; contains lipid expose PPS rafts; expose PPSMajor Tetraspanins Integrins, CR1 and CD133; no TNFRI Histones protein(e.g., CD63, selectins and proteolytic CD63 markers CD9), Alix, CD40ligand enzymes; no TSG101 CD63 Intracellular Internal Plasma PlasmaPlasma origin compartments membrane membrane membrane (endosomes)Abbreviations: phosphatidylserine (PPS); electron microscopy (EM)

Vesicles include shed membrane bound particles, or “microparticles,”that are derived from either the plasma membrane or an internalmembrane. Vesicles can be released into the extracellular environmentfrom cells. Cells releasing vesicles include without limitation cellsthat originate from, or are derived from, the ectoderm, endoderm, ormesoderm. The cells may have undergone genetic, environmental, and/orany other variations or alterations. For example, the cell can be tumorcells. A vesicle can reflect any changes in the source cell, and therebyreflect changes in the originating cells, e.g., cells having variousgenetic mutations. In one mechanism, a vesicle is generatedintracellularly when a segment of the cell membrane spontaneouslyinvaginates and is ultimately exocytosed (see for example, Keller etal., Immunol. Lett. 107 (2): 102-8 (2006)). Vesicles also includecell-derived structures bounded by a lipid bilayer membrane arising fromboth herniated evagination (blebbing) separation and sealing of portionsof the plasma membrane or from the export of any intracellularmembrane-bounded vesicular structure containing variousmembrane-associated proteins of tumor origin, including surface-boundmolecules derived from the host circulation that bind selectively to thetumor-derived proteins together with molecules contained in the vesiclelumen, including but not limited to tumor-derived microRNAs orintracellular proteins. Blebs and blebbing are further described inCharras et al., Nature Reviews Molecular and Cell Biology, Vol. 9, No.11, p. 730-736 (2008). A vesicle shed into circulation or bodily fluidsfrom tumor cells may be referred to as a “circulating tumor-derivedvesicle.” When such vesicle is an exosome, it may be referred to as acirculating-tumor derived exosome (CTE). In some instances, a vesiclecan be derived from a specific cell of origin. CTE, as with acell-of-origin specific vesicle, typically have one or more uniquebiomarkers that permit isolation of the CTE or cell-of-origin specificvesicle, e.g., from a bodily fluid and sometimes in a specific manner.For example, a cell or tissue specific markers are used to identify thecell of origin. Examples of such cell or tissue specific markers aredisclosed herein and can further be accessed in the Tissue-specific GeneExpression and Regulation (TiGER) Database, available atbioinfo.wilmer.jhu.edu/tiger/; Liu et al. (2008) TiGER: a database fortissue-specific gene expression and regulation. BMC Bioinformatics.9:271; TissueDistributionDBs, available atgenome.dkfz-heidelberg.de/menu/tissue_db/index.html.

A vesicle can have a diameter of greater than about 10 nm, 20 nm, or 30nm. A vesicle can have a diameter of greater than 40 nm, 50 nm, 100 nm,200 nm, 500 nm, 1000 nm or greater than 10,000 nm. A vesicle can have adiameter of about 30-1000 nm, about 30-800 nm, about 30-200 nm, or about30-100 nm. In some embodiments, the vesicle has a diameter of less than10,000 nm, 1000 nm, 800 nm, 500 nm, 200 nm, 100 nm, 50 nm, 40 nm, 30 nm,20 nm or less than 10 nm. As used herein the term “about” in referenceto a numerical value means that variations of 10% above or below thenumerical value are within the range ascribed to the specified value.Typical sizes for various types of vesicles are shown in Table 1.Vesicles can be assessed to measure the diameter of a single vesicle orany number of vesicles. For example, the range of diameters of a vesiclepopulation or an average diameter of a vesicle population can bedetermined. Vesicle diameter can be assessed using methods known in theart, e.g., imaging technologies such as electron microscopy. In anembodiment, a diameter of one or more vesicles is determined usingoptical particle detection. See, e.g., U.S. Pat. No. 7,751,053, entitled“Optical Detection and Analysis of Particles” and issued Jul. 6, 2010;and U.S. Pat. No. 7,399,600, entitled “Optical Detection and Analysis ofParticles” and issued Jul. 15, 2010.

In some embodiments, vesicles are directly assayed from a biologicalsample without prior isolation, purification, or concentration from thebiological sample. For example, the amount of vesicles in the sample canby itself provide a biosignature that provides a diagnostic, prognosticor theranostic determination. Alternatively, the vesicle in the samplemay be isolated, captured, purified, or concentrated from a sample priorto analysis. As noted, isolation, capture or purification as used hereincomprises partial isolation, partial capture or partial purificationapart from other components in the sample. Vesicle isolation can beperformed using various techniques as described herein or known in theart, including without limitation size exclusion chromatography, densitygradient centrifugation, differential centrifugation, nanomembraneultrafiltration, immunoabsorbent capture, affinity purification,affinity capture, immunoassay, immunoprecipitation, microfluidicseparation, flow cytometry or combinations thereof.

Vesicles can be assessed to provide a phenotypic characterization bycomparing vesicle characteristics to a reference. In some embodiments,surface antigens on a vesicle are assessed. A vesicle or vesiclepopulation carrying a specific marker can be referred to as a positive(biomarker+) vesicle or vesicle population. For example, a DLL4+population refers to a vesicle population associated with DLL4.Conversely, a DLL4− population would not be associated with DLL4. Thesurface antigens can provide an indication of the anatomical originand/or cellular of the vesicles and other phenotypic information, e.g.,tumor status. For example, vesicles found in a patient sample can beassessed for surface antigens indicative of colorectal origin and thepresence of cancer, thereby identifying vesicles associated withcolorectal cancer cells. The surface antigens may comprise anyinformative biological entity that can be detected on the vesiclemembrane surface, including without limitation surface proteins, lipids,carbohydrates, and other membrane components. For example, positivedetection of colon derived vesicles expressing tumor antigens canindicate that the patient has colorectal cancer. As such, methods of theinvention can be used to characterize any disease or conditionassociated with an anatomical or cellular origin, by assessing, forexample, disease-specific and cell-specific biomarkers of one or morevesicles obtained from a subject.

In embodiments, one or more vesicle payloads are assessed to provide aphenotypic characterization. The payload with a vesicle comprises anyinformative biological entity that can be detected as encapsulatedwithin the vesicle, including without limitation proteins and nucleicacids, e.g., genomic or cDNA, mRNA, or functional fragments thereof, aswell as microRNAs (miRs). In addition, methods of the invention aredirected to detecting vesicle surface antigens (in addition or exclusiveto vesicle payload) to provide a phenotypic characterization. Forexample, vesicles can be characterized by using binding agents (e.g.,antibodies or aptamers) that are specific to vesicle surface antigens,and the bound vesicles can be further assessed to identify one or morepayload components disclosed therein. As described herein, the levels ofvesicles with surface antigens of interest or with payload of interestcan be compared to a reference to characterize a phenotype. For example,overexpression in a sample of cancer-related surface antigens or vesiclepayload, e.g., a tumor associated mRNA or microRNA, as compared to areference, can indicate the presence of cancer in the sample. Thebiomarkers assessed can be present or absent, increased or reduced basedon the selection of the desired target sample and comparison of thetarget sample to the desired reference sample. Non-limiting examples oftarget samples include: disease; treated/not-treated; different timepoints, such as a in a longitudinal study; and non-limiting examples ofreference sample: non-disease; normal; different time points; andsensitive or resistant to candidate treatment(s).

In an embodiment, molecular profiling of the invention comprisesanalysis of microvesicles, such as circulating microvesicles.

MicroRNA

Various biomarker molecules can be assessed in biological samples orvesicles obtained from such biological samples. MicroRNAs comprise oneclass biomarkers assessed via methods of the invention. MicroRNAs, alsoreferred to herein as miRNAs or miRs, are short RNA strandsapproximately 21-23 nucleotides in length. MiRNAs are encoded by genesthat are transcribed from DNA but are not translated into protein andthus comprise non-coding RNA. The miRs are processed from primarytranscripts known as pri-miRNA to short stem-loop structures calledpre-miRNA and finally to the resulting single strand miRNA. Thepre-miRNA typically forms a structure that folds back on itself inself-complementary regions. These structures are then processed by thenuclease Dicer in animals or DCL1 in plants. Mature miRNA molecules arepartially complementary to one or more messenger RNA (mRNA) moleculesand can function to regulate translation of proteins. Identifiedsequences of miRNA can be accessed at publicly available databases, suchas www.microRNA.org, www.mirbase.org, orwww.mirz.unibas.ch/cgi/miRNA.cgi.

miRNAs are generally assigned a number according to the namingconvention “mir-[number].” The number of a miRNA is assigned accordingto its order of discovery relative to previously identified miRNAspecies. For example, if the last published miRNA was mir-121, the nextdiscovered miRNA will be named mir-122, etc. When a miRNA is discoveredthat is homologous to a known miRNA from a different organism, the namecan be given an optional organism identifier, of the form [organismidentifier]-mir-[number]. Identifiers include hsa for Homo sapiens andmmu for Mus Musculus. For example, a human homolog to mir-121 might bereferred to as hsa-mir-121 whereas the mouse homolog can be referred toas mmu-mir-121.

Mature microRNA is commonly designated with the prefix “miR” whereas thegene or precursor miRNA is designated with the prefix “mir.” Forexample, mir-121 is a precursor for miR-121. When differing miRNA genesor precursors are processed into identical mature miRNAs, thegenes/precursors can be delineated by a numbered suffix. For example,mir-121-1 and mir-121-2 can refer to distinct genes or precursors thatare processed into miR-121. Lettered suffixes are used to indicateclosely related mature sequences. For example, mir-121a and mir-121b canbe processed to closely related miRNAs miR-121a and miR-121b,respectively. In the context of the invention, any microRNA (miRNA ormiR) designated herein with the prefix mir-* or miR-* is understood toencompass both the precursor and/or mature species, unless otherwiseexplicitly stated otherwise.

Sometimes it is observed that two mature miRNA sequences originate fromthe same precursor. When one of the sequences is more abundant that theother, a “*” suffix can be used to designate the less common variant.For example, miR-121 would be the predominant product whereas miR-121*is the less common variant found on the opposite arm of the precursor.If the predominant variant is not identified, the miRs can bedistinguished by the suffix “5p” for the variant from the 5′ arm of theprecursor and the suffix “3p” for the variant from the 3′ arm. Forexample, miR-121-5p originates from the 5′ arm of the precursor whereasmiR-121-3p originates from the 3′ arm. Less commonly, the 5p and 3 pvariants are referred to as the sense (“s”) and anti-sense (“as”) forms,respectively. For example, miR-121-5p may be referred to as miR-121-swhereas miR-121-3p may be referred to as miR-121-as.

The above naming conventions have evolved over time and are generalguidelines rather than absolute rules. For example, the let- andlin-families of miRNAs continue to be referred to by these monikers. Themir/miR convention for precursor/mature forms is also a guideline andcontext should be taken into account to determine which form is referredto. Further details of miR naming can be found at www.mirbase.org orAmbros et al., A uniform system for microRNA annotation, RNA 9:277-279(2003).

Plant miRNAs follow a different naming convention as described in Meyerset al., Plant Cell. 2008 20(12):3186-3190.

A number of miRNAs are involved in gene regulation, and miRNAs are partof a growing class of non-coding RNAs that is now recognized as a majortier of gene control. In some cases, miRNAs can interrupt translation bybinding to regulatory sites embedded in the 3′-UTRs of their targetmRNAs, leading to the repression of translation. Target recognitioninvolves complementary base pairing of the target site with the miRNA'sseed region (positions 2-8 at the miRNA's 5′ end), although the exactextent of seed complementarity is not precisely determined and can bemodified by 3′ pairing. In other cases, miRNAs function like smallinterfering RNAs (siRNA) and bind to perfectly complementary mRNAsequences to destroy the target transcript.

Characterization of a number of miRNAs indicates that they influence avariety of processes, including early development, cell proliferationand cell death, apoptosis and fat metabolism. For example, some miRNAs,such as lin-4, let-7, mir-14, mir-23, and bantam, have been shown toplay critical roles in cell differentiation and tissue development.Others are believed to have similarly important roles because of theirdifferential spatial and temporal expression patterns.

The miRNA database available at miRBase (www.mirbase.org) comprises asearchable database of published miRNA sequences and annotation. Furtherinformation about miRBase can be found in the following articles, eachof which is incorporated by reference in its entirety herein:Griffiths-Jones et al., miRBase: tools for microRNA genomics. NAR 200836(Database Issue):D154-D158; Griffiths-Jones et al., miRBase: microRNAsequences, targets and gene nomenclature. NAR 2006 34(DatabaseIssue):D140-D144; and Griffiths-Jones, S. The microRNA Registry. NAR2004 32(Database Issue):D109-D111. Representative miRNAs contained inRelease 16 of miRBase, made available September 2010.

As described herein, microRNAs are known to be involved in cancer andother diseases and can be assessed in order to characterize a phenotypein a sample. See, e.g., Ferracin et al., Micromarkers: miRNAs in cancerdiagnosis and prognosis, Exp Rev Mol Diag, April 2010, Vol. 10, No. 3,Pages 297-308; Fabbri, miRNAs as molecular biomarkers of cancer, Exp RevMol Diag, May 2010, Vol. 10, No. 4, Pages 435-444.

In an embodiment, molecular profiling of the invention comprisesanalysis of microRNA.

Techniques to isolate and characterize vesicles and miRs are known tothose of skill in the art. In addition to the methodology presentedherein, additional methods can be found in U.S. Pat. No. 7,888,035,entitled “METHODS FOR ASSESSING RNA PATTERNS” and issued Feb. 15, 2011;and U.S. Pat. No. 7,897,356, entitled “METHODS AND SYSTEMS OF USINGEXOSOMES FOR DETERMINING PHENOTYPES” and issued Mar. 1, 2011; andInternational Patent Publication Nos. WO/2011/066589, entitled “METHODSAND SYSTEMS FOR ISOLATING, STORING, AND ANALYZING VESICLES” and filedNov. 30, 2010; WO/2011/088226, entitled “DETECTION OF GASTROINTESTINALDISORDERS” and filed Jan. 13, 2011; WO/2011/109440, entitled “BIOMARKERSFOR THERANOSTICS” and filed Mar. 1, 2011; and WO/2011/127219, entitled“CIRCULATING BIOMARKERS FOR DISEASE” and filed Apr. 6, 2011, each ofwhich applications are incorporated by reference herein in theirentirety.

Circulating Biomarkers

Circulating biomarkers include biomarkers that are detectable in bodyfluids, such as blood, plasma, serum. Examples of circulating cancerbiomarkers include cardiac troponin T (cTnT), prostate specific antigen(PSA) for prostate cancer and CA125 for ovarian cancer. Circulatingbiomarkers according to the invention include any appropriate biomarkerthat can be detected in bodily fluid, including without limitationprotein, nucleic acids, e.g., DNA, mRNA and microRNA, lipids,carbohydrates and metabolites. Circulating biomarkers can includebiomarkers that are not associated with cells, such as biomarkers thatare membrane associated, embedded in membrane fragments, part of abiological complex, or free in solution. In one embodiment, circulatingbiomarkers are biomarkers that are associated with one or more vesiclespresent in the biological fluid of a subject.

Circulating biomarkers have been identified for use in characterizationof various phenotypes, such as detection of a cancer. See, e.g., AhmedN, et al., Proteomic-based identification of haptoglobin-1 precursor asa novel circulating biomarker of ovarian cancer. Br. J. Cancer 2004;Mathelin et al., Circulating proteinic biomarkers and breast cancer,Gynecol Obstet Fertil. 2006 July-August; 34(7-8):638-46. Epub 2006 Jul.28; Ye et al., Recent technical strategies to identify diagnosticbiomarkers for ovarian cancer. Expert Rev Proteomics. 2007 February;4(1):121-31; Carney, Circulating oncoproteins HER2/neu, EGFR and CAIX(MN) as novel cancer biomarkers. Expert Rev Mol Diagn. 2007 May;7(3):309-19; Gagnon, Discovery and application of protein biomarkers forovarian cancer, Curr Opin Obstet Gynecol. 2008 February; 20(1):9-13;Pasterkamp et al., Immune regulatory cells: circulating biomarkerfactories in cardiovascular disease. Clin Sci (Lond). 2008 August;115(4):129-31; Fabbri, miRNAs as molecular biomarkers of cancer, Exp RevMol Diag, May 2010, Vol. 10, No. 4, Pages 435-444; PCT PatentPublication WO/2007/088537; U.S. Pat. Nos. 7,745,150 and 7,655,479; U.S.Patent Publications 20110008808, 20100330683, 20100248290, 20100222230,20100203566, 20100173788, 20090291932, 20090239246, 20090226937,20090111121, 20090004687, 20080261258, 20080213907, 20060003465,20050124071, and 20040096915, each of which publication is incorporatedherein by reference in its entirety. In an embodiment, molecularprofiling of the invention comprises analysis of circulating biomarkers.

Gene Expression Profiling

The methods and systems of the invention comprise expression profiling,which includes assessing differential expression of one or more targetgenes disclosed herein. Differential expression can includeoverexpression and/or underexpression of a biological product, e.g., agene, mRNA or protein, compared to a control (or a reference). Thecontrol can include similar cells to the sample but without the disease(e.g., expression profiles obtained from samples from healthyindividuals). A control can be a previously determined level that isindicative of a drug target efficacy associated with the particulardisease and the particular drug target. The control can be derived fromthe same patient, e.g., a normal adjacent portion of the same organ asthe diseased cells, the control can be derived from healthy tissues fromother patients, or previously determined thresholds that are indicativeof a disease responding or not-responding to a particular drug target.The control can also be a control found in the same sample, e.g. ahousekeeping gene or a product thereof (e.g., mRNA or protein). Forexample, a control nucleic acid can be one which is known not to differdepending on the cancerous or non-cancerous state of the cell. Theexpression level of a control nucleic acid can be used to normalizesignal levels in the test and reference populations. Illustrativecontrol genes include, but are not limited to, e.g., β-actin,glyceraldehyde 3-phosphate dehydrogenase and ribosomal protein P1.Multiple controls or types of controls can be used. The source ofdifferential expression can vary. For example, a gene copy number may beincreased in a cell, thereby resulting in increased expression of thegene. Alternately, transcription of the gene may be modified, e.g., bychromatin remodeling, differential methylation, differential expressionor activity of transcription factors, etc. Translation may also bemodified, e.g., by differential expression of factors that degrade mRNA,translate mRNA, or silence translation, e.g., microRNAs or siRNAs. Insome embodiments, differential expression comprises differentialactivity. For example, a protein may carry a mutation that increases theactivity of the protein, such as constitutive activation, therebycontributing to a diseased state. Molecular profiling that revealschanges in activity can be used to guide treatment selection.

Methods of gene expression profiling include methods based onhybridization analysis of polynucleotides, and methods based onsequencing of polynucleotides. Commonly used methods known in the artfor the quantification of mRNA expression in a sample include northernblotting and in situ hybridization (Parker & Barnes (1999) Methods inMolecular Biology 106:247-283); RNAse protection assays (Hod (1992)Biotechniques 13:852-854); and reverse transcription polymerase chainreaction (RT-PCR) (Weis et al. (1992) Trends in Genetics 8:263-264).Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. Representative methods forsequencing-based gene expression analysis include Serial Analysis ofGene Expression (SAGE), gene expression analysis by massively parallelsignature sequencing (MPSS) and/or next generation sequencing.

RT-PCR

Reverse transcription polymerase chain reaction (RT-PCR) is a variant ofpolymerase chain reaction (PCR). According to this technique, a RNAstrand is reverse transcribed into its DNA complement (i.e.,complementary DNA, or cDNA) using the enzyme reverse transcriptase, andthe resulting cDNA is amplified using PCR. Real-time polymerase chainreaction is another PCR variant, which is also referred to asquantitative PCR, Q-PCR, qRT-PCR, or sometimes as RT-PCR. Either thereverse transcription PCR method or the real-time PCR method can be usedfor molecular profiling according to the invention, and RT-PCR can referto either unless otherwise specified or as understood by one of skill inthe art.

RT-PCR can be used to determine RNA levels, e.g., mRNA or miRNA levels,of the biomarkers of the invention. RT-PCR can be used to compare suchRNA levels of the biomarkers of the invention in different samplepopulations, in normal and tumor tissues, with or without drugtreatment, to characterize patterns of gene expression, to discriminatebetween closely related RNAs, and to analyze RNA structure.

The first step is the isolation of RNA, e.g., mRNA, from a sample. Thestarting material can be total RNA isolated from human tumors or tumorcell lines, and corresponding normal tissues or cell lines,respectively. Thus RNA can be isolated from a sample, e.g., tumor cellsor tumor cell lines, and compared with pooled DNA from healthy donors.If the source of mRNA is a primary tumor, mRNA can be extracted, forexample, from frozen or archived paraffin-embedded and fixed (e.g.formalin-fixed) tissue samples.

General methods for mRNA extraction are well known in the art and aredisclosed in standard textbooks of molecular biology, including Ausubelet al. (1997) Current Protocols of Molecular Biology, John Wiley andSons. Methods for RNA extraction from paraffin embedded tissues aredisclosed, for example, in Rupp & Locker (1987) Lab Invest. 56:A67, andDe Andres et al., BioTechniques 18:42044 (1995). In particular, RNAisolation can be performed using purification kit, buffer set andprotease from commercial manufacturers, such as Qiagen, according to themanufacturer's instructions (QIAGEN Inc., Valencia, Calif.). Forexample, total RNA from cells in culture can be isolated using QiagenRNeasy mini-columns. Numerous RNA isolation kits are commerciallyavailable and can be used in the methods of the invention.

In the alternative, the first step is the isolation of miRNA from atarget sample. The starting material is typically total RNA isolatedfrom human tumors or tumor cell lines, and corresponding normal tissuesor cell lines, respectively. Thus RNA can be isolated from a variety ofprimary tumors or tumor cell lines, with pooled DNA from healthy donors.If the source of miRNA is a primary tumor, miRNA can be extracted, forexample, from frozen or archived paraffin-embedded and fixed (e.g.formalin-fixed) tissue samples.

General methods for miRNA extraction are well known in the art and aredisclosed in standard textbooks of molecular biology, including Ausubelet al. (1997) Current Protocols of Molecular Biology, John Wiley andSons. Methods for RNA extraction from paraffin embedded tissues aredisclosed, for example, in Rupp & Locker (1987) Lab Invest. 56:A67, andDe Andres et al., BioTechniques 18:42044 (1995). In particular, RNAisolation can be performed using purification kit, buffer set andprotease from commercial manufacturers, such as Qiagen, according to themanufacturer's instructions. For example, total RNA from cells inculture can be isolated using Qiagen RNeasy mini-columns. Numerous miRNAisolation kits are commercially available and can be used in the methodsof the invention.

Whether the RNA comprises mRNA, miRNA or other types of RNA, geneexpression profiling by RT-PCR can include reverse transcription of theRNA template into cDNA, followed by amplification in a PCR reaction.Commonly used reverse transcriptases include, but are not limited to,avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloneymurine leukemia virus reverse transcriptase (MMLV-RT). The reversetranscription step is typically primed using specific primers, randomhexamers, or oligo-dT primers, depending on the circumstances and thegoal of expression profiling. For example, extracted RNA can bereverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, Calif.,USA), following the manufacturer's instructions. The derived cDNA canthen be used as a template in the subsequent PCR reaction.

Although the PCR step can use a variety of thermostable DNA-dependentDNA polymerases, it typically employs the Taq DNA polymerase, which hasa 5′-3′ nuclease activity but lacks a 3′-5′ proofreading endonucleaseactivity. TaqMan PCR typically uses the 5′-nuclease activity of Taq orTth polymerase to hydrolyze a hybridization probe bound to its targetamplicon, but any enzyme with equivalent 5′ nuclease activity can beused. Two oligonucleotide primers are used to generate an amplicontypical of a PCR reaction. A third oligonucleotide, or probe, isdesigned to detect nucleotide sequence located between the two PCRprimers. The probe is non-extendible by Taq DNA polymerase enzyme, andis labeled with a reporter fluorescent dye and a quencher fluorescentdye. Any laser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

TaqMan™ RT-PCR can be performed using commercially available equipment,such as, for example, ABI PRISM 7700™ Sequence Detection System™(Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), orLightCycler (Roche Molecular Biochemicals, Mannheim, Germany). In onespecific embodiment, the 5′ nuclease procedure is run on a real-timequantitative PCR device such as the ABI PRISM 7700 Sequence DetectionSystem. The system consists of a thermocycler, laser, charge-coupleddevice (CCD), camera and computer. The system amplifies samples in a96-well format on a thermocycler. During amplification, laser-inducedfluorescent signal is collected in real-time through fiber optic cablesfor all 96 wells, and detected at the CCD. The system includes softwarefor running the instrument and for analyzing the data.

TaqMan data are initially expressed as Ct, or the threshold cycle. Asdiscussed above, fluorescence values are recorded during every cycle andrepresent the amount of product amplified to that point in theamplification reaction. The point when the fluorescent signal is firstrecorded as statistically significant is the threshold cycle (Ct).

To minimize errors and the effect of sample-to-sample variation, RT-PCRis usually performed using an internal standard. The ideal internalstandard is expressed at a constant level among different tissues, andis unaffected by the experimental treatment. RNAs most frequently usedto normalize patterns of gene expression are mRNAs for the housekeepinggenes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin.

Real time quantitative PCR (also quantitative real time polymerase chainreaction, QRT-PCR or Q-PCR) is a more recent variation of the RT-PCRtechnique. Q-PCR can measure PCR product accumulation through adual-labeled fluorigenic probe (i.e., TaqMan probe). Real time PCR iscompatible both with quantitative competitive PCR, where internalcompetitor for each target sequence is used for normalization, and withquantitative comparative PCR using a normalization gene contained withinthe sample, or a housekeeping gene for RT-PCR. See, e.g. Held et al.(1996) Genome Research 6:986-994.

Protein-based detection techniques are also useful for molecularprofiling, especially when the nucleotide variant causes amino acidsubstitutions or deletions or insertions or frame shift that affect theprotein primary, secondary or tertiary structure. To detect the aminoacid variations, protein sequencing techniques may be used. For example,a protein or fragment thereof corresponding to a gene can be synthesizedby recombinant expression using a DNA fragment isolated from anindividual to be tested. Preferably, a cDNA fragment of no more than 100to 150 base pairs encompassing the polymorphic locus to be determined isused. The amino acid sequence of the peptide can then be determined byconventional protein sequencing methods. Alternatively, theHPLC-microscopy tandem mass spectrometry technique can be used fordetermining the amino acid sequence variations. In this technique,proteolytic digestion is performed on a protein, and the resultingpeptide mixture is separated by reversed-phase chromatographicseparation. Tandem mass spectrometry is then performed and the datacollected is analyzed. See Gatlin et al., Anal. Chem., 72:757-763(2000).

Microarray

The biomarkers of the invention can also be identified, confirmed,and/or measured using the microarray technique. Thus, the expressionprofile biomarkers can be measured in cancer samples using microarraytechnology. In this method, polynucleotide sequences of interest areplated, or arrayed, on a microchip substrate. The arrayed sequences arethen hybridized with specific DNA probes from cells or tissues ofinterest. The source of mRNA can be total RNA isolated from a sample,e.g., human tumors or tumor cell lines and corresponding normal tissuesor cell lines. Thus RNA can be isolated from a variety of primary tumorsor tumor cell lines. If the source of mRNA is a primary tumor, mRNA canbe extracted, for example, from frozen or archived paraffin-embedded andfixed (e.g. formalin-fixed) tissue samples, which are routinely preparedand preserved in everyday clinical practice.

The expression profile of biomarkers can be measured in either fresh orparaffin-embedded tumor tissue, or body fluids using microarraytechnology. In this method, polynucleotide sequences of interest areplated, or arrayed, on a microchip substrate. The arrayed sequences arethen hybridized with specific DNA probes from cells or tissues ofinterest. As with the RT-PCR method, the source of miRNA typically istotal RNA isolated from human tumors or tumor cell lines, including bodyfluids, such as serum, urine, tears, and exosomes and correspondingnormal tissues or cell lines. Thus RNA can be isolated from a variety ofsources. If the source of miRNA is a primary tumor, miRNA can beextracted, for example, from frozen tissue samples, which are routinelyprepared and preserved in everyday clinical practice.

Also known as biochip, DNA chip, or gene array, cDNA microarraytechnology allows for identification of gene expression levels in abiologic sample. cDNAs or oligonucleotides, each representing a givengene, are immobilized on a substrate, e.g., a small chip, bead or nylonmembrane, tagged, and serve as probes that will indicate whether theyare expressed in biologic samples of interest. The simultaneousexpression of thousands of genes can be monitored simultaneously.

In a specific embodiment of the microarray technique, PCR amplifiedinserts of cDNA clones are applied to a substrate in a dense array. Inone aspect, at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000,1,500, 2,000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000,20,000, 25,000, 30,000, 35,000, 40,000, 45,000 or at least 50,000nucleotide sequences are applied to the substrate. Each sequence cancorrespond to a different gene, or multiple sequences can be arrayed pergene. The microarrayed genes, immobilized on the microchip, are suitablefor hybridization under stringent conditions. Fluorescently labeled cDNAprobes may be generated through incorporation of fluorescent nucleotidesby reverse transcription of RNA extracted from tissues of interest.Labeled cDNA probes applied to the chip hybridize with specificity toeach spot of DNA on the array. After stringent washing to removenon-specifically bound probes, the chip is scanned by confocal lasermicroscopy or by another detection method, such as a CCD camera.Quantitation of hybridization of each arrayed element allows forassessment of corresponding mRNA abundance. With dual colorfluorescence, separately labeled cDNA probes generated from two sourcesof RNA are hybridized pairwise to the array. The relative abundance ofthe transcripts from the two sources corresponding to each specifiedgene is thus determined simultaneously. The miniaturized scale of thehybridization affords a convenient and rapid evaluation of theexpression pattern for large numbers of genes. Such methods have beenshown to have the sensitivity required to detect rare transcripts, whichare expressed at a few copies per cell, and to reproducibly detect atleast approximately two-fold differences in the expression levels(Schena et al. (1996) Proc. Natl. Acad. Sci. USA 93(2):106-149).Microarray analysis can be performed by commercially available equipmentfollowing manufacturer's protocols, including without limitation theAffymetrix GeneChip technology (Affymetrix, Santa Clara, Calif.),Agilent (Agilent Technologies, Inc., Santa Clara, Calif.), or Illumina(Illumina, Inc., San Diego, Calif.) microarray technology.

The development of microarray methods for large-scale analysis of geneexpression makes it possible to search systematically for molecularmarkers of cancer classification and outcome prediction in a variety oftumor types.

In some embodiments, the Agilent Whole Human Genome Microarray Kit(Agilent Technologies, Inc., Santa Clara, Calif.). The system cananalyze more than 41,000 unique human genes and transcripts represented,all with public domain annotations. The system is used according to themanufacturer's instructions.

In some embodiments, the Illumina Whole Genome DASL assay (IlluminaInc., San Diego, Calif.) is used. The system offers a method tosimultaneously profile over 24,000 transcripts from minimal RNA input,from both fresh frozen (FF) and formalin-fixed paraffin embedded (FFPE)tissue sources, in a high throughput fashion.

Microarray expression analysis comprises identifying whether a gene orgene product is up-regulated or down-regulated relative to a reference.The identification can be performed using a statistical test todetermine statistical significance of any differential expressionobserved. In some embodiments, statistical significance is determinedusing a parametric statistical test. The parametric statistical test cancomprise, for example, a fractional factorial design, analysis ofvariance (ANOVA), a t-test, least squares, a Pearson correlation, simplelinear regression, nonlinear regression, multiple linear regression, ormultiple nonlinear regression. Alternatively, the parametric statisticaltest can comprise a one-way analysis of variance, two-way analysis ofvariance, or repeated measures analysis of variance. In otherembodiments, statistical significance is determined using anonparametric statistical test. Examples include, but are not limitedto, a Wilcoxon signed-rank test, a Mann-Whitney test, a Kruskal-Wallistest, a Friedman test, a Spearman ranked order correlation coefficient,a Kendall Tau analysis, and a nonparametric regression test. In someembodiments, statistical significance is determined at a p-value of lessthan about 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001. Although themicroarray systems used in the methods of the invention may assaythousands of transcripts, data analysis need only be performed on thetranscripts of interest, thereby reducing the problem of multiplecomparisons inherent in performing multiple statistical tests. Thep-values can also be corrected for multiple comparisons, e.g., using aBonferroni correction, a modification thereof, or other technique knownto those in the art, e.g., the Hochberg correction, Holm-Bonferronicorrection, Šidák correction, or Dunnett's correction. The degree ofdifferential expression can also be taken into account. For example, agene can be considered as differentially expressed when the fold-changein expression compared to control level is at least 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, 4, 5, 6, 7, 8, 9 or 10-folddifferent in the sample versus the control. The differential expressiontakes into account both overexpression and underexpression. A gene orgene product can be considered up or down-regulated if the differentialexpression meets a statistical threshold, a fold-change threshold, orboth. For example, the criteria for identifying differential expressioncan comprise both a p-value of 0.001 and fold change of at least1.5-fold (up or down). One of skill will understand that suchstatistical and threshold measures can be adapted to determinedifferential expression by any molecular profiling technique disclosedherein.

Various methods of the invention make use of many types of microarraysthat detect the presence and potentially the amount of biologicalentities in a sample. Arrays typically contain addressable moieties thatcan detect the presence of the entity in the sample, e.g., via a bindingevent. Microarrays include without limitation DNA microarrays, such ascDNA microarrays, oligonucleotide microarrays and SNP microarrays,microRNA arrays, protein microarrays, antibody microarrays, tissuemicroarrays, cellular microarrays (also called transfectionmicroarrays), chemical compound microarrays, and carbohydrate arrays(glycoarrays). DNA arrays typically comprise addressable nucleotidesequences that can bind to sequences present in a sample. MicroRNAarrays, e.g., the MMChips array from the University of Louisville orcommercial systems from Agilent, can be used to detect microRNAs.Protein microarrays can be used to identify protein—proteininteractions, including without limitation identifying substrates ofprotein kinases, transcription factor protein-activation, or to identifythe targets of biologically active small molecules. Protein arrays maycomprise an array of different protein molecules, commonly antibodies,or nucleotide sequences that bind to proteins of interest. Antibodymicroarrays comprise antibodies spotted onto the protein chip that areused as capture molecules to detect proteins or other biologicalmaterials from a sample, e.g., from cell or tissue lysate solutions. Forexample, antibody arrays can be used to detect biomarkers from bodilyfluids, e.g., serum or urine, for diagnostic applications. Tissuemicroarrays comprise separate tissue cores assembled in array fashion toallow multiplex histological analysis. Cellular microarrays, also calledtransfection microarrays, comprise various capture agents, such asantibodies, proteins, or lipids, which can interact with cells tofacilitate their capture on addressable locations. Chemical compoundmicroarrays comprise arrays of chemical compounds and can be used todetect protein or other biological materials that bind the compounds.Carbohydrate arrays (glycoarrays) comprise arrays of carbohydrates andcan detect, e.g., protein that bind sugar moieties. One of skill willappreciate that similar technologies or improvements can be usedaccording to the methods of the invention.

Certain embodiments of the current methods comprise a multi-wellreaction vessel, including without limitation, a multi-well plate or amulti-chambered microfluidic device, in which a multiplicity ofamplification reactions and, in some embodiments, detection areperformed, typically in parallel. In certain embodiments, one or moremultiplex reactions for generating amplicons are performed in the samereaction vessel, including without limitation, a multi-well plate, suchas a 96-well, a 384-well, a 1536-well plate, and so forth; or amicrofluidic device, for example but not limited to, a TaqMan™ LowDensity Array (Applied Biosystems, Foster City, Calif.). In someembodiments, a massively parallel amplifying step comprises a multi-wellreaction vessel, including a plate comprising multiple reaction wells,for example but not limited to, a 24-well plate, a 96-well plate, a384-well plate, or a 1536-well plate; or a multi-chamber microfluidicsdevice, for example but not limited to a low density array wherein eachchamber or well comprises an appropriate primer(s), primer set(s),and/or reporter probe(s), as appropriate. Typically such amplificationsteps occur in a series of parallel single-plex, two-plex, three-plex,four-plex, five-plex, or six-plex reactions, although higher levels ofparallel multiplexing are also within the intended scope of the currentteachings. These methods can comprise PCR methodology, such as RT-PCR,in each of the wells or chambers to amplify and/or detect nucleic acidmolecules of interest.

Low density arrays can include arrays that detect 10s or 100s ofmolecules as opposed to 1000s of molecules. These arrays can be moresensitive than high density arrays. In embodiments, a low density arraysuch as a TaqMan™ Low Density Array is used to detect one or more geneor gene product in any of Table 2, Tables 6-9 or Tables 12-15. Forexample, the low density array can be used to detect at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100genes or gene products in Table 2, Tables 6-9 or Tables 12-15.

In some embodiments, the disclosed methods comprise a microfluidicsdevice, “lab on a chip,” or micrototal analytical system (pTAS). In someembodiments, sample preparation is performed using a microfluidicsdevice. In some embodiments, an amplification reaction is performedusing a microfluidics device. In some embodiments, a sequencing or PCRreaction is performed using a microfluidic device. In some embodiments,the nucleotide sequence of at least a part of an amplified product isobtained using a microfluidics device. In some embodiments, detectingcomprises a microfluidic device, including without limitation, a lowdensity array, such as a TaqMan™ Low Density Array. Descriptions ofexemplary microfluidic devices can be found in, among other places,Published PCT Application Nos. WO/0185341 and WO 04/011666; Kartalov andQuake, Nucl. Acids Res. 32:2873-79, 2004; and Fiorini and Chiu, BioTechniques 38:429-46, 2005.

Any appropriate microfluidic device can be used in the methods of theinvention. Examples of microfluidic devices that may be used, or adaptedfor use with molecular profiling, include but are not limited to thosedescribed in U.S. Pat. Nos. 7,591,936, 7,581,429, 7,579,136, 7,575,722,7,568,399, 7,552,741, 7,544,506, 7,541,578, 7,518,726, 7,488,596,7,485,214, 7,467,928, 7,452,713, 7,452,509, 7,449,096, 7,431,887,7,422,725, 7,422,669, 7,419,822, 7,419,639, 7,413,709, 7,411,184,7,402,229, 7,390,463, 7,381,471, 7,357,864, 7,351,592, 7,351,380,7,338,637, 7,329,391, 7,323,140, 7,261,824, 7,258,837, 7,253,003,7,238,324, 7,238,255, 7,233,865, 7,229,538, 7,201,881, 7,195,986,7,189,581, 7,189,580, 7,189,368, 7,141,978, 7,138,062, 7,135,147,7,125,711, 7,118,910, 7,118,661, 7,640,947, 7,666,361, 7,704,735; U.S.Patent Application Publication 20060035243; and International PatentPublication WO 2010/072410; each of which patents or applications areincorporated herein by reference in their entirety. Another example foruse with methods disclosed herein is described in Chen et al.,“Microfluidic isolation and transcriptome analysis of serum vesicles,”Lab on a Chip, Dec. 8, 2009 DOI: 10. 1039/b916199f.

Gene Expression Analysis by Massively Parallel Signature Sequencing(MPSS)

This method, described by Brenner et al. (2000) Nature Biotechnology18:630-634, is a sequencing approach that combines non-gel-basedsignature sequencing with in vitro cloning of millions of templates onseparate microbeads. First, a microbead library of DNA templates isconstructed by in vitro cloning. This is followed by the assembly of aplanar array of the template-containing microbeads in a flow cell at ahigh density. The free ends of the cloned templates on each microbeadare analyzed simultaneously, using a fluorescence-based signaturesequencing method that does not require DNA fragment separation. Thismethod has been shown to simultaneously and accurately provide, in asingle operation, hundreds of thousands of gene signature sequences froma cDNA library.

MPSS data has many uses. The expression levels of nearly all transcriptscan be quantitatively determined; the abundance of signatures isrepresentative of the expression level of the gene in the analyzedtissue. Quantitative methods for the analysis of tag frequencies anddetection of differences among libraries have been published andincorporated into public databases for SAGE™ data and are applicable toMPSS data. The availability of complete genome sequences permits thedirect comparison of signatures to genomic sequences and further extendsthe utility of MPSS data. Because the targets for MPSS analysis are notpre-selected (like on a microarray), MPSS data can characterize the fullcomplexity of transcriptomes. This is analogous to sequencing millionsof ESTs at once, and genomic sequence data can be used so that thesource of the MPSS signature can be readily identified by computationalmeans.

Serial Analysis of Gene Expression (SAGE)

Serial analysis of gene expression (SAGE) is a method that allows thesimultaneous and quantitative analysis of a large number of genetranscripts, without the need of providing an individual hybridizationprobe for each transcript. First, a short sequence tag (e.g., about10-14 bp) is generated that contains sufficient information to uniquelyidentify a transcript, provided that the tag is obtained from a uniqueposition within each transcript. Then, many transcripts are linkedtogether to form long serial molecules, that can be sequenced, revealingthe identity of the multiple tags simultaneously. The expression patternof any population of transcripts can be quantitatively evaluated bydetermining the abundance of individual tags, and identifying the genecorresponding to each tag. See, e.g. Velculescu et al. (1995) Science270:484-487; and Velculescu et al. (1997) Cell 88:243-51.

DNA Copy Number Profiling

Any method capable of determining a DNA copy number profile of aparticular sample can be used for molecular profiling according to theinvention as long as the resolution is sufficient to identify thebiomarkers of the invention. The skilled artisan is aware of and capableof using a number of different platforms for assessing whole genome copynumber changes at a resolution sufficient to identify the copy number ofthe one or more biomarkers of the invention. Some of the platforms andtechniques are described in the embodiments below. In some embodimentsof the invention, ISH techniques as described herein are also used fordetermining copy number/gene amplification.

In some embodiments, the copy number profile analysis involvesamplification of whole genome DNA by a whole genome amplificationmethod. The whole genome amplification method can use a stranddisplacing polymerase and random primers.

In some aspects of these embodiments, the copy number profile analysisinvolves hybridization of whole genome amplified DNA with a high densityarray. In a more specific aspect, the high density array has 5,000 ormore different probes. In another specific aspect, the high densityarray has 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 300,000,400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 ormore different probes. In another specific aspect, each of the differentprobes on the array is an oligonucleotide having from about 15 to 200bases in length. In another specific aspect, each of the differentprobes on the array is an oligonucleotide having from about 15 to 200,15 to 150, 15 to 100, 15 to 75, 15 to 60, or 20 to 55 bases in length.

In some embodiments, a microarray is employed to aid in determining thecopy number profile for a sample, e.g., cells from a tumor. Microarraystypically comprise a plurality of oligomers (e.g., DNA or RNApolynucleotides or oligonucleotides, or other polymers), synthesized ordeposited on a substrate (e.g., glass support) in an array pattern. Thesupport-bound oligomers are “probes”, which function to hybridize orbind with a sample material (e.g., nucleic acids prepared or obtainedfrom the tumor samples), in hybridization experiments. The reversesituation can also be applied: the sample can be bound to the microarraysubstrate and the oligomer probes are in solution for the hybridization.In use, the array surface is contacted with one or more targets underconditions that promote specific, high-affinity binding of the target toone or more of the probes. In some configurations, the sample nucleicacid is labeled with a detectable label, such as a fluorescent tag, sothat the hybridized sample and probes are detectable with scanningequipment. DNA array technology offers the potential of using amultitude (e.g., hundreds of thousands) of different oligonucleotides toanalyze DNA copy number profiles. In some embodiments, the substratesused for arrays are surface-derivatized glass or silica, or polymermembrane surfaces (see e.g., in Z. Guo, et al., Nucleic Acids Res, 22,5456-65 (1994); U. Maskos, E. M. Southern, Nucleic Acids Res, 20,1679-84 (1992), and E. M. Southern, et al., Nucleic Acids Res, 22,1368-73 (1994), each incorporated by reference herein). Modification ofsurfaces of array substrates can be accomplished by many techniques. Forexample, siliceous or metal oxide surfaces can be derivatized withbifunctional silanes, i.e., silanes having a first functional groupenabling covalent binding to the surface (e.g., Si-halogen or Si-alkoxygroup, as in —SiCl₃ or —Si(OCH₃)₃, respectively) and a second functionalgroup that can impart the desired chemical and/or physical modificationsto the surface to covalently or non-covalently attach ligands and/or thepolymers or monomers for the biological probe array. Silylatedderivatizations and other surface derivatizations that are known in theart (see for example U.S. Pat. No. 5,624,711 to Sundberg, U.S. Pat. No.5,266,222 to Willis, and U.S. Pat. No. 5,137,765 to Farnsworth, eachincorporated by reference herein). Other processes for preparing arraysare described in U.S. Pat. No. 6,649,348, to Bass et. al., assigned toAgilent Corp., which disclose DNA arrays created by in situ synthesismethods.

Polymer array synthesis is also described extensively in the literatureincluding in the following: WO 00/58516, U.S. Pat. Nos. 5,143,854,5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,424,186,5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639,5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716,5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324, 5,968,740,5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193,6,090,555, 6,136,269, 6,269,846 and 6,428,752, 5,412,087, 6,147,205,6,262,216, 6,310,189, 5,889,165, and 5,959,098 in PCT Applications Nos.PCT/US99/00730 (International Publication No. WO 99/36760) andPCT/US01/04285 (International Publication No. WO 01/58593), which areall incorporated herein by reference in their entirety for all purposes.

Nucleic acid arrays that are useful in the present invention include,but are not limited to, those that are commercially available fromAffymetrix (Santa Clara, Calif.) under the brand name GeneChip™ Examplearrays are shown on the website at affymetrix.com. Another microarraysupplier is Illumina, Inc., of San Diego, Calif. with example arraysshown on their website at illumina.com.

In some embodiments, the inventive methods provide for samplepreparation. Depending on the microarray and experiment to be performed,sample nucleic acid can be prepared in a number of ways by methods knownto the skilled artisan. In some aspects of the invention, prior to orconcurrent with genotyping (analysis of copy number profiles), thesample may be amplified any number of mechanisms. The most commonamplification procedure used involves PCR. See, for example, PCRTechnology: Principles and Applications for DNA Amplification (Ed. H. A.Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide toMethods and Applications (Eds. Innis, et al., Academic Press, San Diego,Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991);Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (Eds.McPherson et al., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202,4,683,195, 4,800,159 4,965,188, and 5,333,675, and each of which isincorporated herein by reference in their entireties for all purposes.In some embodiments, the sample may be amplified on the array (e.g.,U.S. Pat. No. 6,300,070 which is incorporated herein by reference)

Other suitable amplification methods include the ligase chain reaction(LCR) (for example, Wu and Wallace, Genomics 4, 560 (1989), Landegren etal., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88/10315), self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat.Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporatedherein by reference). Other amplification methods that may be used aredescribed in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S.Ser. No. 09/854,317, each of which is incorporated herein by reference.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al., GenomeResearch 11, 1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592 andU.S. Ser. Nos. 09/916,135, 09/920,491 (U.S. Patent ApplicationPublication 20030096235), Ser. No. 09/910,292 (U.S. Patent ApplicationPublication 20030082543), and Ser. No. 10/013,598.

Methods for conducting polynucleotide hybridization assays are welldeveloped in the art. Hybridization assay procedures and conditions usedin the methods of the invention will vary depending on the applicationand are selected in accordance with the general binding methods knownincluding those referred to in: Maniatis et al. Molecular Cloning: ALaboratory Manual (2.sup.nd Ed. Cold Spring Harbor, N.Y., 1989); Bergerand Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular CloningTechniques (Academic Press, Inc., San Diego, Calif., 1987); Young andDavism, P.N.A.S, 80: 1194 (1983). Methods and apparatus for carrying outrepeated and controlled hybridization reactions have been described inU.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623each of which are incorporated herein by reference.

The methods of the invention may also involve signal detection ofhybridization between ligands in after (and/or during) hybridization.See U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758;5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639;6,218,803; and 6,225,625, in U.S. Ser. No. 10/389,194 and in PCTApplication PCT/US99/06097 (published as WO99/47964), each of which alsois hereby incorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. Nos. 10/389,194,60/493,495 and in PCT Application PCT/US99/06097 (published asWO99/47964), each of which also is hereby incorporated by reference inits entirety for all purposes.

Immuno-Based Assays

Protein-based detection molecular profiling techniques includeimmunoaffinity assays based on antibodies selectively immunoreactivewith mutant gene encoded protein according to the present invention.These techniques include without limitation immunoprecipitation, Westernblot analysis, molecular binding assays, enzyme-linked immunosorbentassay (ELISA), enzyme-linked immunofiltration assay (ELIFA),fluorescence activated cell sorting (FACS) and the like. For example, anoptional method of detecting the expression of a biomarker in a samplecomprises contacting the sample with an antibody against the biomarker,or an immunoreactive fragment of the antibody thereof, or a recombinantprotein containing an antigen binding region of an antibody against thebiomarker; and then detecting the binding of the biomarker in thesample. Methods for producing such antibodies are known in the art.Antibodies can be used to immunoprecipitate specific proteins fromsolution samples or to immunoblot proteins separated by, e.g.,polyacrylamide gels. Immunocytochemical methods can also be used indetecting specific protein polymorphisms in tissues or cells. Otherwell-known antibody-based techniques can also be used including, e.g.,ELISA, radioimmunoassay (RIA), immunoradiometric assays (IRMA) andimmunoenzymatic assays (IEMA), including sandwich assays usingmonoclonal or polyclonal antibodies. See, e.g., U.S. Pat. Nos. 4,376,110and 4,486,530, both of which are incorporated herein by reference.

In alternative methods, the sample may be contacted with an antibodyspecific for a biomarker under conditions sufficient for anantibody-biomarker complex to form, and then detecting said complex. Thepresence of the biomarker may be detected in a number of ways, such asby Western blotting and ELISA procedures for assaying a wide variety oftissues and samples, including plasma or serum. A wide range ofimmunoassay techniques using such an assay format are available, see,e.g., U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These includeboth single-site and two-site or “sandwich” assays of thenon-competitive types, as well as in the traditional competitive bindingassays. These assays also include direct binding of a labelled antibodyto a target biomarker.

A number of variations of the sandwich assay technique exist, and allare intended to be encompassed by the present invention. Briefly, in atypical forward assay, an unlabelled antibody is immobilized on a solidsubstrate, and the sample to be tested brought into contact with thebound molecule. After a suitable period of incubation, for a period oftime sufficient to allow formation of an antibody-antigen complex, asecond antibody specific to the antigen, labelled with a reportermolecule capable of producing a detectable signal is then added andincubated, allowing time sufficient for the formation of another complexof antibody-antigen-labelled antibody. Any unreacted material is washedaway, and the presence of the antigen is determined by observation of asignal produced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample containing known amountsof biomarker.

Variations on the forward assay include a simultaneous assay, in whichboth sample and labelled antibody are added simultaneously to the boundantibody. These techniques are well known to those skilled in the art,including any minor variations as will be readily apparent. In a typicalforward sandwich assay, a first antibody having specificity for thebiomarker is either covalently or passively bound to a solid surface.The solid surface is typically glass or a polymer, the most commonlyused polymers being cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene. The solid supports may be in theform of tubes, beads, discs of microplates, or any other surfacesuitable for conducting an immunoassay. The binding processes arewell-known in the art and generally consist of cross-linking covalentlybinding or physically adsorbing, the polymer-antibody complex is washedin preparation for the test sample. An aliquot of the sample to betested is then added to the solid phase complex and incubated for aperiod of time sufficient (e.g. 2-40 minutes or overnight if moreconvenient) and under suitable conditions (e.g. from room temperature to40° C. such as between 25° C. and 32° C. inclusive) to allow binding ofany subunit present in the antibody. Following the incubation period,the antibody subunit solid phase is washed and dried and incubated witha second antibody specific for a portion of the biomarker. The secondantibody is linked to a reporter molecule which is used to indicate thebinding of the second antibody to the molecular marker.

An alternative method involves immobilizing the target biomarkers in thesample and then exposing the immobilized target to specific antibodywhich may or may not be labelled with a reporter molecule. Depending onthe amount of target and the strength of the reporter molecule signal, abound target may be detectable by direct labelling with the antibody.Alternatively, a second labelled antibody, specific to the firstantibody is exposed to the target-first antibody complex to form atarget-first antibody-second antibody tertiary complex. The complex isdetected by the signal emitted by the reporter molecule. By “reportermolecule”, as used in the present specification, is meant a moleculewhich, by its chemical nature, provides an analytically identifiablesignal which allows the detection of antigen-bound antibody. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to thesecond antibody, generally by means of glutaraldehyde or periodate. Aswill be readily recognized, however, a wide variety of differentconjugation techniques exist, which are readily available to the skilledartisan. Commonly used enzymes include horseradish peroxidase, glucoseoxidase, β-galactosidase and alkaline phosphatase, amongst others. Thesubstrates to be used with the specific enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable color change. Examples of suitable enzymes include alkalinephosphatase and peroxidase. It is also possible to employ fluorogenicsubstrates, which yield a fluorescent product rather than thechromogenic substrates noted above. In all cases, the enzyme-labelledantibody is added to the first antibody-molecular marker complex,allowed to bind, and then the excess reagent is washed away. A solutioncontaining the appropriate substrate is then added to the complex ofantibody-antigen-antibody. The substrate will react with the enzymelinked to the second antibody, giving a qualitative visual signal, whichmay be further quantitated, usually spectrophotometrically, to give anindication of the amount of biomarker which was present in the sample.Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. As in the EIA, the fluorescent labelled antibody is allowedto bind to the first antibody-molecular marker complex. After washingoff the unbound reagent, the remaining tertiary complex is then exposedto the light of the appropriate wavelength, the fluorescence observedindicates the presence of the molecular marker of interest.Immunofluorescence and EIA techniques are both very well established inthe art. However, other reporter molecules, such as radioisotope,chemiluminescent or bioluminescent molecules, may also be employed.

Immunohistochemistry (IHC)

IHC is a process of localizing antigens (e.g., proteins) in cells of atissue binding antibodies specifically to antigens in the tissues. Theantigen-binding antibody can be conjugated or fused to a tag that allowsits detection, e.g., via visualization. In some embodiments, the tag isan enzyme that can catalyze a color-producing reaction, such as alkalinephosphatase or horseradish peroxidase. The enzyme can be fused to theantibody or non-covalently bound, e.g., using a biotin-avadin system.Alternatively, the antibody can be tagged with a fluorophore, such asfluorescein, rhodamine, DyLight Fluor or Alexa Fluor. Theantigen-binding antibody can be directly tagged or it can itself berecognized by a detection antibody that carries the tag. Using IHC, oneor more proteins may be detected. The expression of a gene product canbe related to its staining intensity compared to control levels. In someembodiments, the gene product is considered differentially expressed ifits staining varies at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.2, 2.5, 2.7, 3.0, 4, 5, 6, 7, 8, 9 or 10-fold in the sampleversus the control.

IHC comprises the application of antigen-antibody interactions tohistochemical techniques. In an illustrative example, a tissue sectionis mounted on a slide and is incubated with antibodies (polyclonal ormonoclonal) specific to the antigen (primary reaction). Theantigen-antibody signal is then amplified using a second antibodyconjugated to a complex of peroxidase antiperoxidase (PAP),avidin-biotin-peroxidase (ABC) or avidin-biotin alkaline phosphatase. Inthe presence of substrate and chromogen, the enzyme forms a coloreddeposit at the sites of antibody-antigen binding. Immunofluorescence isan alternate approach to visualize antigens. In this technique, theprimary antigen-antibody signal is amplified using a second antibodyconjugated to a fluorochrome. On UV light absorption, the fluorochromeemits its own light at a longer wavelength (fluorescence), thus allowinglocalization of antibody-antigen complexes.

Epigenetic Status

Molecular profiling methods according to the invention also comprisemeasuring epigenetic change, i.e., modification in a gene caused by anepigenetic mechanism, such as a change in methylation status or histoneacetylation. Frequently, the epigenetic change will result in analteration in the levels of expression of the gene which may be detected(at the RNA or protein level as appropriate) as an indication of theepigenetic change. Often the epigenetic change results in silencing ordown regulation of the gene, referred to as “epigenetic silencing.” Themost frequently investigated epigenetic change in the methods of theinvention involves determining the DNA methylation status of a gene,where an increased level of methylation is typically associated with therelevant cancer (since it may cause down regulation of gene expression).Aberrant methylation, which may be referred to as hypermethylation, ofthe gene or genes can be detected. Typically, the methylation status isdetermined in suitable CpG islands which are often found in the promoterregion of the gene(s). The term “methylation,” “methylation state” or“methylation status” may refers to the presence or absence of5-methylcytosine at one or a plurality of CpG dinucleotides within a DNAsequence. CpG dinucleotides are typically concentrated in the promoterregions and exons of human genes.

Diminished gene expression can be assessed in terms of DNA methylationstatus or in terms of expression levels as determined by the methylationstatus of the gene. One method to detect epigenetic silencing is todetermine that a gene which is expressed in normal cells is lessexpressed or not expressed in tumor cells. Accordingly, the inventionprovides for a method of molecular profiling comprising detectingepigenetic silencing.

Various assay procedures to directly detect methylation are known in theart, and can be used in conjunction with the present invention. Theseassays rely onto two distinct approaches: bisulphite conversion basedapproaches and non-bisulphite based approaches. Non-bisulphite basedmethods for analysis of DNA methylation rely on the inability ofmethylation-sensitive enzymes to cleave methylation cytosines in theirrestriction. The bisulphite conversion relies on treatment of DNAsamples with sodium bisulphite which converts unmethylated cytosine touracil, while methylated cytosines are maintained (Furuichi Y, Wataya Y,Hayatsu H, Ukita T. Biochem Biophys Res Commun. 1970 Dec. 9;41(5):1185-91). This conversion results in a change in the sequence ofthe original DNA. Methods to detect such changes include MS AP-PCR(Methylation-Sensitive Arbitrarily-Primed Polymerase Chain Reaction), atechnology that allows for a global scan of the genome using CG-richprimers to focus on the regions most likely to contain CpGdinucleotides, and described by Gonzalgo et al., Cancer Research57:594-599, 1997; MethyLight™, which refers to the art-recognizedfluorescence-based real-time PCR technique described by Eads et al.,Cancer Res. 59:2302-2306, 1999; the HeavyMethylTMassay, in theembodiment thereof implemented herein, is an assay, wherein methylationspecific blocking probes (also referred to herein as blockers) coveringCpG positions between, or covered by the amplification primers enablemethylation-specific selective amplification of a nucleic acid sample;HeavyMethylTMMethyLight™ is a variation of the MethyLight™ assay whereinthe MethyLight™ assay is combined with methylation specific blockingprobes covering CpG positions between the amplification primers;Ms-SNuPE (Methylation-sensitive Single Nucleotide Primer Extension) isan assay described by Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531,1997; MSP (Methylation-specific PCR) is a methylation assay described byHerman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996, and by U.S.Pat. No. 5,786,146; COBRA (Combined Bisulfite Restriction Analysis) is amethylation assay described by Xiong & Laird, Nucleic Acids Res.25:2532-2534, 1997; MCA (Methylated CpG Island Amplification) is amethylation assay described by Toyota et al., Cancer Res. 59:2307-12,1999, and in WO 00/26401A1.

Other techniques for DNA methylation analysis include sequencing,methylation-specific PCR (MS-PCR), melting curve methylation-specificPCR (McMS-PCR), MLPA with or without bisulfite treatment, QAMA,MSRE-PCR, MethyLight, ConLight-MSP, bisulfite conversion-specificmethylation-specific PCR (BS-MSP), COBRA (which relies upon use ofrestriction enzymes to reveal methylation dependent sequence differencesin PCR products of sodium bisulfite-treated DNA), methylation-sensitivesingle-nucleotide primer extension conformation (MS-SNuPE),methylation-sensitive single-strand conformation analysis (MS-SSCA),Melting curve combined bisulfite restriction analysis (McCOBRA),PyroMethA, HeavyMethyl, MALDI-TOF, MassARRAY, Quantitative analysis ofmethylated alleles (QAMA), enzymatic regional methylation assay (ERMA),QBSUPT, MethylQuant, Quantitative PCR sequencing andoligonucleotide-based microarray systems, Pyrosequencing, Meth-DOP-PCR.A review of some useful techniques is provided in Nucleic acidsresearch, 1998, Vol. 26, No. 10, 2255-2264; Nature Reviews, 2003, Vol.3, 253-266; Oral Oncology, 2006, Vol. 42, 5-13, which references areincorporated herein in their entirety. Any of these techniques may beused in accordance with the present invention, as appropriate. Othertechniques are described in U.S. Patent Publications 20100144836; and20100184027, which applications are incorporated herein by reference intheir entirety.

Through the activity of various acetylases and deacetylylases the DNAbinding function of histone proteins is tightly regulated. Furthermore,histone acetylation and histone deactelyation have been linked withmalignant progression. See Nature, 429: 457-63, 2004. Methods to analyzehistone acetylation are described in U.S. Patent Publications20100144543 and 20100151468, which applications are incorporated hereinby reference in their entirety.

Sequence Analysis

Molecular profiling according to the present invention comprises methodsfor genotyping one or more biomarkers by determining whether anindividual has one or more nucleotide variants (or amino acid variants)in one or more of the genes or gene products. Genotyping one or moregenes according to the methods of the invention in some embodiments, canprovide more evidence for selecting a treatment.

The biomarkers of the invention can be analyzed by any method useful fordetermining alterations in nucleic acids or the proteins they encode.According to one embodiment, the ordinary skilled artisan can analyzethe one or more genes for mutations including deletion mutants,insertion mutants, frame shift mutants, nonsense mutants, missensemutant, and splice mutants.

Nucleic acid used for analysis of the one or more genes can be isolatedfrom cells in the sample according to standard methodologies (Sambrooket al., 1989). The nucleic acid, for example, may be genomic DNA orfractionated or whole cell RNA, or miRNA acquired from exosomes or cellsurfaces. Where RNA is used, it may be desired to convert the RNA to acomplementary DNA. In one embodiment, the RNA is whole cell RNA; inanother, it is poly-A RNA; in another, it is exosomal RNA. Normally, thenucleic acid is amplified. Depending on the format of the assay foranalyzing the one or more genes, the specific nucleic acid of interestis identified in the sample directly using amplification or with asecond, known nucleic acid following amplification. Next, the identifiedproduct is detected. In certain applications, the detection may beperformed by visual means (e.g., ethidium bromide staining of a gel).Alternatively, the detection may involve indirect identification of theproduct via chemiluminescence, radioactive scintigraphy of radiolabel orfluorescent label or even via a system using electrical or thermalimpulse signals (Affymax Technology; Bellus, 1994).

Various types of defects are known to occur in the biomarkers of theinvention. Alterations include without limitation deletions, insertions,point mutations, and duplications. Point mutations can be silent or canresult in stop codons, frame shift mutations or amino acidsubstitutions. Mutations in and outside the coding region of the one ormore genes may occur and can be analyzed according to the methods of theinvention. The target site of a nucleic acid of interest can include theregion wherein the sequence varies. Examples include, but are notlimited to, polymorphisms which exist in different forms such as singlenucleotide variations, nucleotide repeats, multibase deletion (more thanone nucleotide deleted from the consensus sequence), multibase insertion(more than one nucleotide inserted from the consensus sequence),microsatellite repeats (small numbers of nucleotide repeats with atypical 5-1000 repeat units), di-nucleotide repeats, tri-nucleotiderepeats, sequence rearrangements (including translocation andduplication), chimeric sequence (two sequences from different geneorigins are fused together), and the like. Among sequence polymorphisms,the most frequent polymorphisms in the human genome are single-basevariations, also called single-nucleotide polymorphisms (SNPs). SNPs areabundant, stable and widely distributed across the genome.

Molecular profiling includes methods for haplotyping one or more genes.The haplotype is a set of genetic determinants located on a singlechromosome and it typically contains a particular combination of alleles(all the alternative sequences of a gene) in a region of a chromosome.In other words, the haplotype is phased sequence information onindividual chromosomes. Very often, phased SNPs on a chromosome define ahaplotype. A combination of haplotypes on chromosomes can determine agenetic profile of a cell. It is the haplotype that determines a linkagebetween a specific genetic marker and a disease mutation. Haplotypingcan be done by any methods known in the art. Common methods of scoringSNPs include hybridization microarray or direct gel sequencing, reviewedin Landgren et al., Genome Research, 8:769-776, 1998. For example, onlyone copy of one or more genes can be isolated from an individual and thenucleotide at each of the variant positions is determined.Alternatively, an allele specific PCR or a similar method can be used toamplify only one copy of the one or more genes in an individual, and theSNPs at the variant positions of the present invention are determined.The Clark method known in the art can also be employed for haplotyping.A high throughput molecular haplotyping method is also disclosed in Tostet al., Nucleic Acids Res., 30(19):e96 (2002), which is incorporatedherein by reference.

Thus, additional variant(s) that are in linkage disequilibrium with thevariants and/or haplotypes of the present invention can be identified bya haplotyping method known in the art, as will be apparent to a skilledartisan in the field of genetics and haplotyping. The additionalvariants that are in linkage disequilibrium with a variant or haplotypeof the present invention can also be useful in the various applicationsas described below.

For purposes of genotyping and haplotyping, both genomic DNA andmRNA/cDNA can be used, and both are herein referred to generically as“gene.”

Numerous techniques for detecting nucleotide variants are known in theart and can all be used for the method of this invention. The techniquescan be protein-based or nucleic acid-based. In either case, thetechniques used must be sufficiently sensitive so as to accuratelydetect the small nucleotide or amino acid variations. Very often, aprobe is used which is labeled with a detectable marker. Unlessotherwise specified in a particular technique described below, anysuitable marker known in the art can be used, including but not limitedto, radioactive isotopes, fluorescent compounds, biotin which isdetectable using streptavidin, enzymes (e.g., alkaline phosphatase),substrates of an enzyme, ligands and antibodies, etc. See Jablonski etal., Nucleic Acids Res., 14:6115-6128 (1986); Nguyen et al.,Biotechniques, 13:116-123 (1992); Rigby et al., J. Mol. Biol.,113:237-251 (1977).

In a nucleic acid-based detection method, target DNA sample, i.e., asample containing genomic DNA, cDNA, mRNA and/or miRNA, corresponding tothe one or more genes must be obtained from the individual to be tested.Any tissue or cell sample containing the genomic DNA, miRNA, mRNA,and/or cDNA (or a portion thereof) corresponding to the one or moregenes can be used. For this purpose, a tissue sample containing cellnucleus and thus genomic DNA can be obtained from the individual. Bloodsamples can also be useful except that only white blood cells and otherlymphocytes have cell nucleus, while red blood cells are without anucleus and contain only mRNA or miRNA. Nevertheless, miRNA and mRNA arealso useful as either can be analyzed for the presence of nucleotidevariants in its sequence or serve as template for cDNA synthesis. Thetissue or cell samples can be analyzed directly without much processing.Alternatively, nucleic acids including the target sequence can beextracted, purified, and/or amplified before they are subject to thevarious detecting procedures discussed below. Other than tissue or cellsamples, cDNAs or genomic DNAs from a cDNA or genomic DNA libraryconstructed using a tissue or cell sample obtained from the individualto be tested are also useful.

To determine the presence or absence of a particular nucleotide variant,sequencing of the target genomic DNA or cDNA, particularly the regionencompassing the nucleotide variant locus to be detected. Varioussequencing techniques are generally known and widely used in the artincluding the Sanger method and Gilbert chemical method. Thepyrosequencing method monitors DNA synthesis in real time using aluminometric detection system. Pyrosequencing has been shown to beeffective in analyzing genetic polymorphisms such as single-nucleotidepolymorphisms and can also be used in the present invention. SeeNordstrom et al., Biotechnol. Appl. Biochem., 31(2):107-112 (2000);Ahmadian et al., Anal. Biochem., 280:103-110 (2000).

Nucleic acid variants can be detected by a suitable detection process.Non limiting examples of methods of detection, quantification,sequencing and the like are; mass detection of mass modified amplicons(e.g., matrix-assisted laser desorption ionization (MALDI) massspectrometry and electrospray (ES) mass spectrometry), a primerextension method (e.g., iPLEX™; Sequenom, Inc.), microsequencing methods(e.g., a modification of primer extension methodology), ligase sequencedetermination methods (e.g., U.S. Pat. Nos. 5,679,524 and 5,952,174, andWO 01/27326), mismatch sequence determination methods (e.g., U.S. Pat.Nos. 5,851,770; 5,958,692; 6,110,684; and 6,183,958), direct DNAsequencing, fragment analysis (FA), restriction fragment lengthpolymorphism (RFLP analysis), allele specific oligonucleotide (ASO)analysis, methylation-specific PCR (MSPCR), pyrosequencing analysis,acycloprime analysis, Reverse dot blot, GeneChip microarrays, Dynamicallele-specific hybridization (DASH), Peptide nucleic acid (PNA) andlocked nucleic acids (LNA) probes, TaqMan, Molecular Beacons,Intercalating dye, FRET primers, AlphaScreen, SNPstream, genetic bitanalysis (GBA), Multiplex minisequencing, SNaPshot, GOOD assay,Microarray miniseq, arrayed primer extension (APEX), Microarray primerextension (e.g., microarray sequence determination methods), Tag arrays,Coded microspheres, Template-directed incorporation (TDI), fluorescencepolarization, Colorimetric oligonucleotide ligation assay (OLA),Sequence-coded OLA, Microarray ligation, Ligase chain reaction, Padlockprobes, Invader assay, hybridization methods (e.g., hybridization usingat least one probe, hybridization using at least one fluorescentlylabeled probe, and the like), conventional dot blot analyses, singlestrand conformational polymorphism analysis (SSCP, e.g., U.S. Pat. Nos.5,891,625 and 6,013,499; Orita et al., Proc. Natl. Acad. Sci. U.S.A. 86:27776-2770 (1989)), denaturing gradient gel electrophoresis (DGGE),heteroduplex analysis, mismatch cleavage detection, and techniquesdescribed in Sheffield et al., Proc. Natl. Acad. Sci. USA 49: 699-706(1991), White et al., Genomics 12: 301-306 (1992), Grompe et al., Proc.Natl. Acad. Sci. USA 86: 5855-5892 (1989), and Grompe, Nature Genetics5: 111-117 (1993), cloning and sequencing, electrophoresis, the use ofhybridization probes and quantitative real time polymerase chainreaction (QRT-PCR), digital PCR, nanopore sequencing, chips andcombinations thereof. The detection and quantification of alleles orparalogs can be carried out using the “closed-tube” methods described inU.S. patent application Ser. No. 11/950,395, filed on Dec. 4, 2007. Insome embodiments the amount of a nucleic acid species is determined bymass spectrometry, primer extension, sequencing (e.g., any suitablemethod, for example nanopore or pyrosequencing), Quantitative PCR (Q-PCRor QRT-PCR), digital PCR, combinations thereof, and the like.

The term “sequence analysis” as used herein refers to determining anucleotide sequence, e.g., that of an amplification product. The entiresequence or a partial sequence of a polynucleotide, e.g., DNA or mRNA,can be determined, and the determined nucleotide sequence can bereferred to as a “read” or “sequence read.” For example, linearamplification products may be analyzed directly without furtheramplification in some embodiments (e.g., by using single-moleculesequencing methodology). In certain embodiments, linear amplificationproducts may be subject to further amplification and then analyzed(e.g., using sequencing by ligation or pyrosequencing methodology).Reads may be subject to different types of sequence analysis. Anysuitable sequencing method can be used to detect, and determine theamount of, nucleotide sequence species, amplified nucleic acid species,or detectable products generated from the foregoing. Examples of certainsequencing methods are described hereafter.

A sequence analysis apparatus or sequence analysis component(s) includesan apparatus, and one or more components used in conjunction with suchapparatus, that can be used by a person of ordinary skill to determine anucleotide sequence resulting from processes described herein (e.g.,linear and/or exponential amplification products). Examples ofsequencing platforms include, without limitation, the 454 platform(Roche) (Margulies, M. et al. 2005 Nature 437, 376-380), IlluminaGenomic Analyzer (or Solexa platform) or SOLID System (AppliedBiosystems; see PCT patent application publications WO 06/084132entitled “Reagents, Methods, and Libraries For Bead-Based Sequencing”and WO07/121,489 entitled “Reagents, Methods, and Libraries for Gel-FreeBead-Based Sequencing”), the Helicos True Single Molecule DNA sequencingtechnology (Harris T D et al. 2008 Science, 320, 106-109), the singlemolecule, real-time (SMRT™) technology of Pacific Biosciences, andnanopore sequencing (Soni G V and Meller A. 2007 Clin Chem 53:1996-2001), Ion semiconductor sequencing (Ion Torrent Systems, Inc, SanFrancisco, Calif.), or DNA nanoball sequencing (Complete Genomics,Mountain View, Calif.), VisiGen Biotechnologies approach (Invitrogen)and polony sequencing. Such platforms allow sequencing of many nucleicacid molecules isolated from a specimen at high orders of multiplexingin a parallel manner (Dear Brief Funct Genomic Proteomic 2003; 1:397-416; Haimovich, Methods, challenges, and promise of next-generationsequencing in cancer biology. Yale J Biol Med. 2011 December;84(4):439-46). These non-Sanger-based sequencing technologies aresometimes referred to as NextGen sequencing, NGS, next-generationsequencing, next generation sequencing, and variations thereof.Typically they allow much higher throughput than the traditional Sangerapproach. See Schuster, Next-generation sequencing transforms today'sbiology, Nature Methods 5:16-18 (2008); Metzker, Sequencingtechnologies—the next generation. Nat Rev Genet. 2010 January;11(1):31-46. These platforms can allow sequencing of clonally expandedor non-amplified single molecules of nucleic acid fragments. Certainplatforms involve, for example, sequencing by ligation of dye-modifiedprobes (including cyclic ligation and cleavage), pyrosequencing, andsingle-molecule sequencing. Nucleotide sequence species, amplificationnucleic acid species and detectable products generated there from can beanalyzed by such sequence analysis platforms. Next-generation sequencingcan be used in the methods of the invention, e.g., to determinemutations, copy number, or expression levels, as appropriate. Themethods can be used to perform whole genome sequencing or sequencing ofspecific sequences of interest, such as a gene of interest or a fragmentthereof.

Sequencing by ligation is a nucleic acid sequencing method that relieson the sensitivity of DNA ligase to base-pairing mismatch. DNA ligasejoins together ends of DNA that are correctly base paired. Combining theability of DNA ligase to join together only correctly base paired DNAends, with mixed pools of fluorescently labeled oligonucleotides orprimers, enables sequence determination by fluorescence detection.Longer sequence reads may be obtained by including primers containingcleavable linkages that can be cleaved after label identification.Cleavage at the linker removes the label and regenerates the 5′phosphate on the end of the ligated primer, preparing the primer foranother round of ligation. In some embodiments primers may be labeledwith more than one fluorescent label, e.g., at least 1, 2, 3, 4, or 5fluorescent labels.

Sequencing by ligation generally involves the following steps. Clonalbead populations can be prepared in emulsion microreactors containingtarget nucleic acid template sequences, amplification reactioncomponents, beads and primers. After amplification, templates aredenatured and bead enrichment is performed to separate beads withextended templates from undesired beads (e.g., beads with no extendedtemplates). The template on the selected beads undergoes a 3′modification to allow covalent bonding to the slide, and modified beadscan be deposited onto a glass slide. Deposition chambers offer theability to segment a slide into one, four or eight chambers during thebead loading process. For sequence analysis, primers hybridize to theadapter sequence. A set of four color dye-labeled probes competes forligation to the sequencing primer. Specificity of probe ligation isachieved by interrogating every 4th and 5th base during the ligationseries. Five to seven rounds of ligation, detection and cleavage recordthe color at every 5th position with the number of rounds determined bythe type of library used. Following each round of ligation, a newcomplimentary primer offset by one base in the 5′ direction is laid downfor another series of ligations. Primer reset and ligation rounds (5-7ligation cycles per round) are repeated sequentially five times togenerate 25-35 base pairs of sequence for a single tag. With mate-pairedsequencing, this process is repeated for a second tag.

Pyrosequencing is a nucleic acid sequencing method based on sequencingby synthesis, which relies on detection of a pyrophosphate released onnucleotide incorporation. Generally, sequencing by synthesis involvessynthesizing, one nucleotide at a time, a DNA strand complimentary tothe strand whose sequence is being sought. Target nucleic acids may beimmobilized to a solid support, hybridized with a sequencing primer,incubated with DNA polymerase, ATP sulfurylase, luciferase, apyrase,adenosine 5′ phosphosulfate and luciferin. Nucleotide solutions aresequentially added and removed. Correct incorporation of a nucleotidereleases a pyrophosphate, which interacts with ATP sulfurylase andproduces ATP in the presence of adenosine 5′ phosphosulfate, fueling theluciferin reaction, which produces a chemiluminescent signal allowingsequence determination. The amount of light generated is proportional tothe number of bases added. Accordingly, the sequence downstream of thesequencing primer can be determined. An illustrative system forpyrosequencing involves the following steps: ligating an adaptor nucleicacid to a nucleic acid under investigation and hybridizing the resultingnucleic acid to a bead; amplifying a nucleotide sequence in an emulsion;sorting beads using a picoliter multiwell solid support; and sequencingamplified nucleotide sequences by pyrosequencing methodology (e.g.,Nakano et al., “Single-molecule PCR using water-in-oil emulsion;”Journal of Biotechnology 102: 117-124 (2003)).

Certain single-molecule sequencing embodiments are based on theprincipal of sequencing by synthesis, and use single-pair FluorescenceResonance Energy Transfer (single pair FRET) as a mechanism by whichphotons are emitted as a result of successful nucleotide incorporation.The emitted photons often are detected using intensified or highsensitivity cooled charge-couple-devices in conjunction with totalinternal reflection microscopy (TIRM). Photons are only emitted when theintroduced reaction solution contains the correct nucleotide forincorporation into the growing nucleic acid chain that is synthesized asa result of the sequencing process. In FRET based single-moleculesequencing, energy is transferred between two fluorescent dyes,sometimes polymethine cyanine dyes Cy3 and Cy5, through long-rangedipole interactions. The donor is excited at its specific excitationwavelength and the excited state energy is transferred, non-radiativelyto the acceptor dye, which in turn becomes excited. The acceptor dyeeventually returns to the ground state by radiative emission of aphoton. The two dyes used in the energy transfer process represent the“single pair” in single pair FRET. Cy3 often is used as the donorfluorophore and often is incorporated as the first labeled nucleotide.Cy5 often is used as the acceptor fluorophore and is used as thenucleotide label for successive nucleotide additions after incorporationof a first Cy3 labeled nucleotide. The fluorophores generally are within10 nanometers of each for energy transfer to occur successfully.

An example of a system that can be used based on single-moleculesequencing generally involves hybridizing a primer to a target nucleicacid sequence to generate a complex; associating the complex with asolid phase; iteratively extending the primer by a nucleotide taggedwith a fluorescent molecule; and capturing an image of fluorescenceresonance energy transfer signals after each iteration (e.g., U.S. Pat.No. 7,169,314; Braslaysky et al., PNAS 100(7): 3960-3964 (2003)). Such asystem can be used to directly sequence amplification products (linearlyor exponentially amplified products) generated by processes describedherein. In some embodiments the amplification products can be hybridizedto a primer that contains sequences complementary to immobilized capturesequences present on a solid support, a bead or glass slide for example.Hybridization of the primer-amplification product complexes with theimmobilized capture sequences, immobilizes amplification products tosolid supports for single pair FRET based sequencing by synthesis. Theprimer often is fluorescent, so that an initial reference image of thesurface of the slide with immobilized nucleic acids can be generated.The initial reference image is useful for determining locations at whichtrue nucleotide incorporation is occurring. Fluorescence signalsdetected in array locations not initially identified in the “primeronly” reference image are discarded as non-specific fluorescence.Following immobilization of the primer-amplification product complexes,the bound nucleic acids often are sequenced in parallel by the iterativesteps of, a) polymerase extension in the presence of one fluorescentlylabeled nucleotide, b) detection of fluorescence using appropriatemicroscopy, TIRM for example, c) removal of fluorescent nucleotide, andd) return to step a with a different fluorescently labeled nucleotide.

In some embodiments, nucleotide sequencing may be by solid phase singlenucleotide sequencing methods and processes. Solid phase singlenucleotide sequencing methods involve contacting target nucleic acid andsolid support under conditions in which a single molecule of samplenucleic acid hybridizes to a single molecule of a solid support. Suchconditions can include providing the solid support molecules and asingle molecule of target nucleic acid in a “microreactor.” Suchconditions also can include providing a mixture in which the targetnucleic acid molecule can hybridize to solid phase nucleic acid on thesolid support. Single nucleotide sequencing methods useful in theembodiments described herein are described in U.S. Provisional PatentApplication Ser. No. 61/021,871 filed Jan. 17, 2008.

In certain embodiments, nanopore sequencing detection methods include(a) contacting a target nucleic acid for sequencing (“base nucleicacid,” e.g., linked probe molecule) with sequence-specific detectors,under conditions in which the detectors specifically hybridize tosubstantially complementary subsequences of the base nucleic acid; (b)detecting signals from the detectors and (c) determining the sequence ofthe base nucleic acid according to the signals detected. In certainembodiments, the detectors hybridized to the base nucleic acid aredisassociated from the base nucleic acid (e.g., sequentiallydissociated) when the detectors interfere with a nanopore structure asthe base nucleic acid passes through a pore, and the detectorsdisassociated from the base sequence are detected. In some embodiments,a detector disassociated from a base nucleic acid emits a detectablesignal, and the detector hybridized to the base nucleic acid emits adifferent detectable signal or no detectable signal. In certainembodiments, nucleotides in a nucleic acid (e.g., linked probe molecule)are substituted with specific nucleotide sequences corresponding tospecific nucleotides (“nucleotide representatives”), thereby giving riseto an expanded nucleic acid (e.g., U.S. Pat. No. 6,723,513), and thedetectors hybridize to the nucleotide representatives in the expandednucleic acid, which serves as a base nucleic acid. In such embodiments,nucleotide representatives may be arranged in a binary or higher orderarrangement (e.g., Soni and Meller, Clinical Chemistry 53(11): 1996-2001(2007)). In some embodiments, a nucleic acid is not expanded, does notgive rise to an expanded nucleic acid, and directly serves a basenucleic acid (e.g., a linked probe molecule serves as a non-expandedbase nucleic acid), and detectors are directly contacted with the basenucleic acid. For example, a first detector may hybridize to a firstsubsequence and a second detector may hybridize to a second subsequence,where the first detector and second detector each have detectable labelsthat can be distinguished from one another, and where the signals fromthe first detector and second detector can be distinguished from oneanother when the detectors are disassociated from the base nucleic acid.In certain embodiments, detectors include a region that hybridizes tothe base nucleic acid (e.g., two regions), which can be about 3 to about100 nucleotides in length (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80,85, 90, or 95 nucleotides in length). A detector also may include one ormore regions of nucleotides that do not hybridize to the base nucleicacid. In some embodiments, a detector is a molecular beacon. A detectoroften comprises one or more detectable labels independently selectedfrom those described herein. Each detectable label can be detected byany convenient detection process capable of detecting a signal generatedby each label (e.g., magnetic, electric, chemical, optical and thelike). For example, a CD camera can be used to detect signals from oneor more distinguishable quantum dots linked to a detector.

In certain sequence analysis embodiments, reads may be used to constructa larger nucleotide sequence, which can be facilitated by identifyingoverlapping sequences in different reads and by using identificationsequences in the reads. Such sequence analysis methods and software forconstructing larger sequences from reads are known to the person ofordinary skill (e.g., Venter et al., Science 291: 1304-1351 (2001)).Specific reads, partial nucleotide sequence constructs, and fullnucleotide sequence constructs may be compared between nucleotidesequences within a sample nucleic acid (i.e., internal comparison) ormay be compared with a reference sequence (i.e., reference comparison)in certain sequence analysis embodiments. Internal comparisons can beperformed in situations where a sample nucleic acid is prepared frommultiple samples or from a single sample source that contains sequencevariations. Reference comparisons sometimes are performed when areference nucleotide sequence is known and an objective is to determinewhether a sample nucleic acid contains a nucleotide sequence that issubstantially similar or the same, or different, than a referencenucleotide sequence. Sequence analysis can be facilitated by the use ofsequence analysis apparatus and components described above.

Primer extension polymorphism detection methods, also referred to hereinas “microsequencing” methods, typically are carried out by hybridizing acomplementary oligonucleotide to a nucleic acid carrying the polymorphicsite. In these methods, the oligonucleotide typically hybridizesadjacent to the polymorphic site. The term “adjacent” as used inreference to “microsequencing” methods, refers to the 3′ end of theextension oligonucleotide being sometimes 1 nucleotide from the 5′ endof the polymorphic site, often 2 or 3, and at times 4, 5, 6, 7, 8, 9, or10 nucleotides from the 5′ end of the polymorphic site, in the nucleicacid when the extension oligonucleotide is hybridized to the nucleicacid. The extension oligonucleotide then is extended by one or morenucleotides, often 1, 2, or 3 nucleotides, and the number and/or type ofnucleotides that are added to the extension oligonucleotide determinewhich polymorphic variant or variants are present. Oligonucleotideextension methods are disclosed, for example, in U.S. Pat. Nos.4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934; 5,908,755;5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431; 6,017,702;6,046,005; 6,087,095; 6,210,891; and WO 01/20039. The extension productscan be detected in any manner, such as by fluorescence methods (see,e.g., Chen & Kwok, Nucleic Acids Research 25: 347-353 (1997) and Chen etal., Proc. Natl. Acad. Sci. USA 94/20: 10756-10761 (1997)) or by massspectrometric methods (e.g., MALDI-TOF mass spectrometry) and othermethods described herein. Oligonucleotide extension methods using massspectrometry are described, for example, in U.S. Pat. Nos. 5,547,835;5,605,798; 5,691,141; 5,849,542; 5,869,242; 5,928,906; 6,043,031;6,194,144; and 6,258,538. Microsequencing detection methods oftenincorporate an amplification process that proceeds the extension step.The amplification process typically amplifies a region from a nucleicacid sample that comprises the polymorphic site. Amplification can becarried out using methods described above, or for example using a pairof oligonucleotide primers in a polymerase chain reaction (PCR), inwhich one oligonucleotide primer typically is complementary to a region3′ of the polymorphism and the other typically is complementary to aregion 5′ of the polymorphism. A PCR primer pair may be used in methodsdisclosed in U.S. Pat. Nos. 4,683,195; 4,683,202, 4,965,188; 5,656,493;5,998,143; 6,140,054; WO 01/27327; and WO 01/27329 for example. PCRprimer pairs may also be used in any commercially available machinesthat perform PCR, such as any of the GeneAmp™ Systems available fromApplied Biosystems.

Other appropriate sequencing methods include multiplex polony sequencing(as described in Shendure et al., Accurate Multiplex Polony Sequencingof an Evolved Bacterial Genome, Sciencexpress, Aug. 4, 2005, pg 1available at www.sciencexpress.org/4 Aug.2005/Pagel/10.1126/science.1117389, incorporated herein by reference),which employs immobilized microbeads, and sequencing in microfabricatedpicoliter reactors (as described in Margulies et al., Genome Sequencingin Microfabricated High-Density Picolitre Reactors, Nature, August 2005,available at www.nature.com/nature (published online 31 Jul. 2005,doi:10.1038/nature03959, incorporated herein by reference).

Whole genome sequencing may also be used for discriminating alleles ofRNA transcripts, in some embodiments. Examples of whole genomesequencing methods include, but are not limited to, nanopore-basedsequencing methods, sequencing by synthesis and sequencing by ligation,as described above.

Nucleic acid variants can also be detected using standardelectrophoretic techniques. Although the detection step can sometimes bepreceded by an amplification step, amplification is not required in theembodiments described herein. Examples of methods for detection andquantification of a nucleic acid using electrophoretic techniques can befound in the art. A non-limiting example comprises running a sample(e.g., mixed nucleic acid sample isolated from maternal serum, oramplification nucleic acid species, for example) in an agarose orpolyacrylamide gel. The gel may be labeled (e.g., stained) with ethidiumbromide (see, Sambrook and Russell, Molecular Cloning: A LaboratoryManual 3d ed., 2001). The presence of a band of the same size as thestandard control is an indication of the presence of a target nucleicacid sequence, the amount of which may then be compared to the controlbased on the intensity of the band, thus detecting and quantifying thetarget sequence of interest. In some embodiments, restriction enzymescapable of distinguishing between maternal and paternal alleles may beused to detect and quantify target nucleic acid species. In certainembodiments, oligonucleotide probes specific to a sequence of interestare used to detect the presence of the target sequence of interest. Theoligonucleotides can also be used to indicate the amount of the targetnucleic acid molecules in comparison to the standard control, based onthe intensity of signal imparted by the probe.

Sequence-specific probe hybridization can be used to detect a particularnucleic acid in a mixture or mixed population comprising other speciesof nucleic acids. Under sufficiently stringent hybridization conditions,the probes hybridize specifically only to substantially complementarysequences. The stringency of the hybridization conditions can be relaxedto tolerate varying amounts of sequence mismatch. A number ofhybridization formats are known in the art, which include but are notlimited to, solution phase, solid phase, or mixed phase hybridizationassays. The following articles provide an overview of the varioushybridization assay formats: Singer et al., Biotechniques 4:230, 1986;Haase et al., Methods in Virology, pp. 189-226, 1984; Wilkinson, In situHybridization, Wilkinson ed., IRL Press, Oxford University Press,Oxford; and Hames and Higgins eds., Nucleic Acid Hybridization: APractical Approach, IRL Press, 1987.

Hybridization complexes can be detected by techniques known in the art.Nucleic acid probes capable of specifically hybridizing to a targetnucleic acid (e.g., mRNA or DNA) can be labeled by any suitable method,and the labeled probe used to detect the presence of hybridized nucleicacids. One commonly used method of detection is autoradiography, usingprobes labeled with ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, or the like. Thechoice of radioactive isotope depends on research preferences due toease of synthesis, stability, and half-lives of the selected isotopes.Other labels include compounds (e.g., biotin and digoxigenin), whichbind to antiligands or antibodies labeled with fluorophores,chemiluminescent agents, and enzymes. In some embodiments, probes can beconjugated directly with labels such as fluorophores, chemiluminescentagents or enzymes. The choice of label depends on sensitivity required,ease of conjugation with the probe, stability requirements, andavailable instrumentation.

In embodiments, fragment analysis (referred to herein as “FA”) methodsare used for molecular profiling. Fragment analysis (FA) includestechniques such as restriction fragment length polymorphism (RFLP)and/or (amplified fragment length polymorphism). If a nucleotide variantin the target DNA corresponding to the one or more genes results in theelimination or creation of a restriction enzyme recognition site, thendigestion of the target DNA with that particular restriction enzyme willgenerate an altered restriction fragment length pattern. Thus, adetected RFLP or AFLP will indicate the presence of a particularnucleotide variant.

Terminal restriction fragment length polymorphism (TRFLP) works by PCRamplification of DNA using primer pairs that have been labeled withfluorescent tags. The PCR products are digested using RFLP enzymes andthe resulting patterns are visualized using a DNA sequencer. The resultsare analyzed either by counting and comparing bands or peaks in theTRFLP profile, or by comparing bands from one or more TRFLP runs in adatabase.

The sequence changes directly involved with an RFLP can also be analyzedmore quickly by PCR. Amplification can be directed across the alteredrestriction site, and the products digested with the restriction enzyme.This method has been called Cleaved Amplified Polymorphic Sequence(CAPS). Alternatively, the amplified segment can be analyzed by Allelespecific oligonucleotide (ASO) probes, a process that is sometimesassessed using a Dot blot.

A variation on AFLP is cDNA-AFLP, which can be used to quantifydifferences in gene expression levels.

Another useful approach is the single-stranded conformation polymorphismassay (SSCA), which is based on the altered mobility of asingle-stranded target DNA spanning the nucleotide variant of interest.A single nucleotide change in the target sequence can result indifferent intramolecular base pairing pattern, and thus differentsecondary structure of the single-stranded DNA, which can be detected ina non-denaturing gel. See Orita et al., Proc. Natl. Acad. Sci. USA,86:2776-2770 (1989). Denaturing gel-based techniques such as clampeddenaturing gel electrophoresis (CDGE) and denaturing gradient gelelectrophoresis (DGGE) detect differences in migration rates of mutantsequences as compared to wild-type sequences in denaturing gel. SeeMiller et al., Biotechniques, 5:1016-24 (1999); Sheffield et al., Am. J.Hum, Genet., 49:699-706 (1991); Wartell et al., Nucleic Acids Res.,18:2699-2705 (1990); and Sheffield et al., Proc. Natl. Acad. Sci. USA,86:232-236 (1989). In addition, the double-strand conformation analysis(DSCA) can also be useful in the present invention. See Arguello et al.,Nat. Genet., 18:192-194 (1998).

The presence or absence of a nucleotide variant at a particular locus inthe one or more genes of an individual can also be detected using theamplification refractory mutation system (ARMS) technique. See e.g.,European Patent No. 0,332,435; Newton et al., Nucleic Acids Res.,17:2503-2515 (1989); Fox et al., Br. J. Cancer, 77:1267-1274 (1998);Robertson et al., Eur. Respir. J., 12:477-482 (1998). In the ARMSmethod, a primer is synthesized matching the nucleotide sequenceimmediately 5′ upstream from the locus being tested except that the3′-end nucleotide which corresponds to the nucleotide at the locus is apredetermined nucleotide. For example, the 3′-end nucleotide can be thesame as that in the mutated locus. The primer can be of any suitablelength so long as it hybridizes to the target DNA under stringentconditions only when its 3′-end nucleotide matches the nucleotide at thelocus being tested. Preferably the primer has at least 12 nucleotides,more preferably from about 18 to 50 nucleotides. If the individualtested has a mutation at the locus and the nucleotide therein matchesthe 3′-end nucleotide of the primer, then the primer can be furtherextended upon hybridizing to the target DNA template, and the primer caninitiate a PCR amplification reaction in conjunction with anothersuitable PCR primer. In contrast, if the nucleotide at the locus is ofwild type, then primer extension cannot be achieved. Various forms ofARMS techniques developed in the past few years can be used. See e.g.,Gibson et al., Clin. Chem. 43:1336-1341 (1997).

Similar to the ARMS technique is the mini sequencing or singlenucleotide primer extension method, which is based on the incorporationof a single nucleotide. An oligonucleotide primer matching thenucleotide sequence immediately 5′ to the locus being tested ishybridized to the target DNA, mRNA or miRNA in the presence of labeleddideoxyribonucleotides. A labeled nucleotide is incorporated or linkedto the primer only when the dideoxyribonucleotides matches thenucleotide at the variant locus being detected. Thus, the identity ofthe nucleotide at the variant locus can be revealed based on thedetection label attached to the incorporated dideoxyribonucleotides. SeeSyvanen et al., Genomics, 8:684-692 (1990); Shumaker et al., Hum.Mutat., 7:346-354 (1996); Chen et al., Genome Res., 10:549-547 (2000).

Another set of techniques useful in the present invention is theso-called “oligonucleotide ligation assay” (OLA) in whichdifferentiation between a wild-type locus and a mutation is based on theability of two oligonucleotides to anneal adjacent to each other on thetarget DNA molecule allowing the two oligonucleotides joined together bya DNA ligase. See Landergren et al., Science, 241:1077-1080 (1988); Chenet al, Genome Res., 8:549-556 (1998); Iannone et al., Cytometry,39:131-140 (2000). Thus, for example, to detect a single-nucleotidemutation at a particular locus in the one or more genes, twooligonucleotides can be synthesized, one having the sequence just 5′upstream from the locus with its 3′ end nucleotide being identical tothe nucleotide in the variant locus of the particular gene, the otherhaving a nucleotide sequence matching the sequence immediately 3′downstream from the locus in the gene. The oligonucleotides can belabeled for the purpose of detection. Upon hybridizing to the targetgene under a stringent condition, the two oligonucleotides are subjectto ligation in the presence of a suitable ligase. The ligation of thetwo oligonucleotides would indicate that the target DNA has a nucleotidevariant at the locus being detected.

Detection of small genetic variations can also be accomplished by avariety of hybridization-based approaches. Allele-specificoligonucleotides are most useful. See Conner et al., Proc. Natl. Acad.Sci. USA, 80:278-282 (1983); Saiki et al, Proc. Natl. Acad. Sci. USA,86:6230-6234 (1989). Oligonucleotide probes (allele-specific)hybridizing specifically to a gene allele having a particular genevariant at a particular locus but not to other alleles can be designedby methods known in the art. The probes can have a length of, e.g., from10 to about 50 nucleotide bases. The target DNA and the oligonucleotideprobe can be contacted with each other under conditions sufficientlystringent such that the nucleotide variant can be distinguished from thewild-type gene based on the presence or absence of hybridization. Theprobe can be labeled to provide detection signals. Alternatively, theallele-specific oligonucleotide probe can be used as a PCR amplificationprimer in an “allele-specific PCR” and the presence or absence of a PCRproduct of the expected length would indicate the presence or absence ofa particular nucleotide variant.

Other useful hybridization-based techniques allow two single-strandednucleic acids annealed together even in the presence of mismatch due tonucleotide substitution, insertion or deletion. The mismatch can then bedetected using various techniques. For example, the annealed duplexescan be subject to electrophoresis. The mismatched duplexes can bedetected based on their electrophoretic mobility that is different fromthe perfectly matched duplexes. See Cariello, Human Genetics, 42:726(1988). Alternatively, in an RNase protection assay, a RNA probe can beprepared spanning the nucleotide variant site to be detected and havinga detection marker. See Giunta et al., Diagn. Mol. Path., 5:265-270(1996); Finkelstein et al., Genomics, 7:167-172 (1990); Kinszler et al.,Science 251:1366-1370 (1991). The RNA probe can be hybridized to thetarget DNA or mRNA forming a heteroduplex that is then subject to theribonuclease RNase A digestion. RNase A digests the RNA probe in theheteroduplex only at the site of mismatch. The digestion can bedetermined on a denaturing electrophoresis gel based on size variations.In addition, mismatches can also be detected by chemical cleavagemethods known in the art. See e.g., Roberts et al., Nucleic Acids Res.,25:3377-3378 (1997).

In the mutS assay, a probe can be prepared matching the gene sequencesurrounding the locus at which the presence or absence of a mutation isto be detected, except that a predetermined nucleotide is used at thevariant locus. Upon annealing the probe to the target DNA to form aduplex, the E. coli mutS protein is contacted with the duplex. Since themutS protein binds only to heteroduplex sequences containing anucleotide mismatch, the binding of the mutS protein will be indicativeof the presence of a mutation. See Modrich et al., Ann. Rev. Genet.,25:229-253 (1991).

A great variety of improvements and variations have been developed inthe art on the basis of the above-described basic techniques which canbe useful in detecting mutations or nucleotide variants in the presentinvention. For example, the “sunrise probes” or “molecular beacons” usethe fluorescence resonance energy transfer (FRET) property and give riseto high sensitivity. See Wolf et al., Proc. Nat. Acad. Sci. USA,85:8790-8794 (1988). Typically, a probe spanning the nucleotide locus tobe detected are designed into a hairpin-shaped structure and labeledwith a quenching fluorophore at one end and a reporter fluorophore atthe other end. In its natural state, the fluorescence from the reporterfluorophore is quenched by the quenching fluorophore due to theproximity of one fluorophore to the other. Upon hybridization of theprobe to the target DNA, the 5′ end is separated apart from the 3′-endand thus fluorescence signal is regenerated. See Nazarenko et al.,Nucleic Acids Res., 25:2516-2521 (1997); Rychlik et al., Nucleic AcidsRes., 17:8543-8551 (1989); Sharkey et al., Bio/Technology 12:506-509(1994); Tyagi et al., Nat. Biotechnol., 14:303-308 (1996); Tyagi et al.,Nat. Biotechnol., 16:49-53 (1998). The homo-tag assisted non-dimersystem (HANDS) can be used in combination with the molecular beaconmethods to suppress primer-dimer accumulation. See Brownie et al.,Nucleic Acids Res., 25:3235-3241 (1997).

Dye-labeled oligonucleotide ligation assay is a FRET-based method, whichcombines the OLA assay and PCR. See Chen et al., Genome Res. 8:549-556(1998). TaqMan is another FRET-based method for detecting nucleotidevariants. A TaqMan probe can be oligonucleotides designed to have thenucleotide sequence of the gene spanning the variant locus of interestand to differentially hybridize with different alleles. The two ends ofthe probe are labeled with a quenching fluorophore and a reporterfluorophore, respectively. The TaqMan probe is incorporated into a PCRreaction for the amplification of a target gene region containing thelocus of interest using Taq polymerase. As Taq polymerase exhibits 5′-3′exonuclease activity but has no 3′-5′ exonuclease activity, if theTaqMan probe is annealed to the target DNA template, the 5′-end of theTaqMan probe will be degraded by Taq polymerase during the PCR reactionthus separating the reporting fluorophore from the quenching fluorophoreand releasing fluorescence signals. See Holland et al., Proc. Natl.Acad. Sci. USA, 88:7276-7280 (1991); Kalinina et al., Nucleic AcidsRes., 25:1999-2004 (1997); Whitcombe et al., Clin. Chem., 44:918-923(1998).

In addition, the detection in the present invention can also employ achemiluminescence-based technique. For example, an oligonucleotide probecan be designed to hybridize to either the wild-type or a variant genelocus but not both. The probe is labeled with a highly chemiluminescentacridinium ester. Hydrolysis of the acridinium ester destroyschemiluminescence. The hybridization of the probe to the target DNAprevents the hydrolysis of the acridinium ester. Therefore, the presenceor absence of a particular mutation in the target DNA is determined bymeasuring chemiluminescence changes. See Nelson et al., Nucleic AcidsRes., 24:4998-5003 (1996).

The detection of genetic variation in the gene in accordance with thepresent invention can also be based on the “base excision sequencescanning” (BESS) technique. The BESS method is a PCR-based mutationscanning method. BESS T-Scan and BESS G-Tracker are generated which areanalogous to T and G ladders of dideoxy sequencing. Mutations aredetected by comparing the sequence of normal and mutant DNA. See, e.g.,Hawkins et al., Electrophoresis, 20:1171-1176 (1999).

Mass spectrometry can be used for molecular profiling according to theinvention. See Graber et al., Curr. Opin. Biotechnol., 9:14-18 (1998).For example, in the primer oligo base extension (PROBE™) method, atarget nucleic acid is immobilized to a solid-phase support. A primer isannealed to the target immediately 5′ upstream from the locus to beanalyzed. Primer extension is carried out in the presence of a selectedmixture of deoxyribonucleotides and dideoxyribonucleotides. Theresulting mixture of newly extended primers is then analyzed byMALDI-TOF. See e.g., Monforte et al., Nat. Med., 3:360-362 (1997).

In addition, the microchip or microarray technologies are alsoapplicable to the detection method of the present invention.Essentially, in microchips, a large number of different oligonucleotideprobes are immobilized in an array on a substrate or carrier, e.g., asilicon chip or glass slide. Target nucleic acid sequences to beanalyzed can be contacted with the immobilized oligonucleotide probes onthe microchip. See Lipshutz et al., Biotechniques, 19:442-447 (1995);Chee et al., Science, 274:610-614 (1996); Kozal et al., Nat. Med.2:753-759 (1996); Hacia et al., Nat. Genet., 14:441-447 (1996); Saiki etal., Proc. Natl. Acad. Sci. USA, 86:6230-6234 (1989); Gingeras et al.,Genome Res., 8:435-448 (1998). Alternatively, the multiple targetnucleic acid sequences to be studied are fixed onto a substrate and anarray of probes is contacted with the immobilized target sequences. SeeDrmanac et al., Nat. Biotechnol., 16:54-58 (1998). Numerous microchiptechnologies have been developed incorporating one or more of the abovedescribed techniques for detecting mutations. The microchip technologiescombined with computerized analysis tools allow fast screening in alarge scale. The adaptation of the microchip technologies to the presentinvention will be apparent to a person of skill in the art apprised ofthe present disclosure. See, e.g., U.S. Pat. No. 5,925,525 to Fodor etal; Wilgenbus et al., J. Mol. Med., 77:761-786 (1999); Graber et al.,Curr. Opin. Biotechnol., 9:14-18 (1998); Hacia et al., Nat. Genet.,14:441-447 (1996); Shoemaker et al., Nat. Genet., 14:450-456 (1996);DeRisi et al., Nat. Genet., 14:457-460 (1996); Chee et al., Nat. Genet.,14:610-614 (1996); Lockhart et al., Nat. Genet., 14:675-680 (1996);Drobyshev et al., Gene, 188:45-52 (1997).

As is apparent from the above survey of the suitable detectiontechniques, it may or may not be necessary to amplify the target DNA,i.e., the gene, cDNA, mRNA, miRNA, or a portion thereof to increase thenumber of target DNA molecule, depending on the detection techniquesused. For example, most PCR-based techniques combine the amplificationof a portion of the target and the detection of the mutations. PCRamplification is well known in the art and is disclosed in U.S. Pat.Nos. 4,683,195 and 4,800,159, both which are incorporated herein byreference. For non-PCR-based detection techniques, if necessary, theamplification can be achieved by, e.g., in vivo plasmid multiplication,or by purifying the target DNA from a large amount of tissue or cellsamples. See generally, Sambrook et al., Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989. However, even with scarce samples, many sensitive techniqueshave been developed in which small genetic variations such assingle-nucleotide substitutions can be detected without having toamplify the target DNA in the sample. For example, techniques have beendeveloped that amplify the signal as opposed to the target DNA by, e.g.,employing branched DNA or dendrimers that can hybridize to the targetDNA. The branched or dendrimer DNAs provide multiple hybridization sitesfor hybridization probes to attach thereto thus amplifying the detectionsignals. See Detmer et al., J. Clin. Microbiol., 34:901-907 (1996);Collins et al., Nucleic Acids Res., 25:2979-2984 (1997); Horn et al.,Nucleic Acids Res., 25:4835-4841 (1997); Horn et al., Nucleic AcidsRes., 25:4842-4849 (1997); Nilsen et al., J. Theor. Biol., 187:273-284(1997).

The Invader™ assay is another technique for detecting single nucleotidevariations that can be used for molecular profiling according to theinvention. The Invader™ assay uses a novel linear signal amplificationtechnology that improves upon the long turnaround times required of thetypical PCR DNA sequenced-based analysis. See Cooksey et al.,Antimicrobial Agents and Chemotherapy 44:1296-1301 (2000). This assay isbased on cleavage of a unique secondary structure formed between twooverlapping oligonucleotides that hybridize to the target sequence ofinterest to form a “flap.” Each “flap” then generates thousands ofsignals per hour. Thus, the results of this technique can be easilyread, and the methods do not require exponential amplification of theDNA target. The Invader™ system uses two short DNA probes, which arehybridized to a DNA target. The structure formed by the hybridizationevent is recognized by a special cleavase enzyme that cuts one of theprobes to release a short DNA “flap.” Each released “flap” then binds toa fluorescently-labeled probe to form another cleavage structure. Whenthe cleavase enzyme cuts the labeled probe, the probe emits a detectablefluorescence signal. See e.g. Lyamichev et al., Nat. Biotechnol.,17:292-296 (1999).

The rolling circle method is another method that avoids exponentialamplification. Lizardi et al., Nature Genetics, 19:225-232 (1998) (whichis incorporated herein by reference). For example, Sniper™, a commercialembodiment of this method, is a sensitive, high-throughput SNP scoringsystem designed for the accurate fluorescent detection of specificvariants. For each nucleotide variant, two linear, allele-specificprobes are designed. The two allele-specific probes are identical withthe exception of the 3′-base, which is varied to complement the variantsite. In the first stage of the assay, target DNA is denatured and thenhybridized with a pair of single, allele-specific, open-circleoligonucleotide probes. When the 3′-base exactly complements the targetDNA, ligation of the probe will preferentially occur. Subsequentdetection of the circularized oligonucleotide probes is by rollingcircle amplification, whereupon the amplified probe products aredetected by fluorescence. See Clark and Pickering, Life Science News 6,2000, Amersham Pharmacia Biotech (2000).

A number of other techniques that avoid amplification all togetherinclude, e.g., surface-enhanced resonance Raman scattering (SERRS),fluorescence correlation spectroscopy, and single-moleculeelectrophoresis. In SERRS, a chromophore-nucleic acid conjugate isabsorbed onto colloidal silver and is irradiated with laser light at aresonant frequency of the chromophore. See Graham et al., Anal. Chem.,69:4703-4707 (1997). The fluorescence correlation spectroscopy is basedon the spatio-temporal correlations among fluctuating light signals andtrapping single molecules in an electric field. See Eigen et al., Proc.Natl. Acad. Sci. USA, 91:5740-5747 (1994). In single-moleculeelectrophoresis, the electrophoretic velocity of a fluorescently taggednucleic acid is determined by measuring the time required for themolecule to travel a predetermined distance between two laser beams. SeeCastro et al., Anal. Chem., 67:3181-3186 (1995).

In addition, the allele-specific oligonucleotides (ASO) can also be usedin in situ hybridization using tissues or cells as samples. Theoligonucleotide probes which can hybridize differentially with thewild-type gene sequence or the gene sequence harboring a mutation may belabeled with radioactive isotopes, fluorescence, or other detectablemarkers. In situ hybridization techniques are well known in the art andtheir adaptation to the present invention for detecting the presence orabsence of a nucleotide variant in the one or more gene of a particularindividual should be apparent to a skilled artisan apprised of thisdisclosure.

Accordingly, the presence or absence of one or more genes nucleotidevariant or amino acid variant in an individual can be determined usingany of the detection methods described above.

Typically, once the presence or absence of one or more gene nucleotidevariants or amino acid variants is determined, physicians or geneticcounselors or patients or other researchers may be informed of theresult. Specifically the result can be cast in a transmittable form thatcan be communicated or transmitted to other researchers or physicians orgenetic counselors or patients. Such a form can vary and can be tangibleor intangible. The result with regard to the presence or absence of anucleotide variant of the present invention in the individual tested canbe embodied in descriptive statements, diagrams, photographs, charts,images or any other visual forms. For example, images of gelelectrophoresis of PCR products can be used in explaining the results.Diagrams showing where a variant occurs in an individual's gene are alsouseful in indicating the testing results. The statements and visualforms can be recorded on a tangible media such as papers, computerreadable media such as floppy disks, compact disks, etc., or on anintangible media, e.g., an electronic media in the form of email orwebsite on internet or intranet. In addition, the result with regard tothe presence or absence of a nucleotide variant or amino acid variant inthe individual tested can also be recorded in a sound form andtransmitted through any suitable media, e.g., analog or digital cablelines, fiber optic cables, etc., via telephone, facsimile, wirelessmobile phone, internet phone and the like.

Thus, the information and data on a test result can be produced anywherein the world and transmitted to a different location. For example, whena genotyping assay is conducted offshore, the information and data on atest result may be generated and cast in a transmittable form asdescribed above. The test result in a transmittable form thus can beimported into the U.S. Accordingly, the present invention alsoencompasses a method for producing a transmittable form of informationon the genotype of the two or more suspected cancer samples from anindividual. The method comprises the steps of (1) determining thegenotype of the DNA from the samples according to methods of the presentinvention; and (2) embodying the result of the determining step in atransmittable form. The transmittable form is the product of theproduction method.

In Situ Hybridization

In situ hybridization assays are well known and are generally describedin Angerer et al., Methods Enzymol. 152:649-660 (1987). In an in situhybridization assay, cells, e.g., from a biopsy, are fixed to a solidsupport, typically a glass slide. If DNA is to be probed, the cells aredenatured with heat or alkali. The cells are then contacted with ahybridization solution at a moderate temperature to permit annealing ofspecific probes that are labeled. The probes are preferably labeled,e.g., with radioisotopes or fluorescent reporters, or enzymatically.FISH (fluorescence in situ hybridization) uses fluorescent probes thatbind to only those parts of a sequence with which they show a highdegree of sequence similarity. CISH (chromogenic in situ hybridization)uses conventional peroxidase or alkaline phosphatase reactionsvisualized under a standard bright-field microscope.

In situ hybridization can be used to detect specific gene sequences intissue sections or cell preparations by hybridizing the complementarystrand of a nucleotide probe to the sequence of interest. Fluorescent insitu hybridization (FISH) uses a fluorescent probe to increase thesensitivity of in situ hybridization.

FISH is a cytogenetic technique used to detect and localize specificpolynucleotide sequences in cells. For example, FISH can be used todetect DNA sequences on chromosomes. FISH can also be used to detect andlocalize specific RNAs, e.g., mRNAs, within tissue samples. In FISH usesfluorescent probes that bind to specific nucleotide sequences to whichthey show a high degree of sequence similarity. Fluorescence microscopycan be used to find out whether and where the fluorescent probes arebound. In addition to detecting specific nucleotide sequences, e.g.,translocations, fusion, breaks, duplications and other chromosomalabnormalities, FISH can help define the spatial-temporal patterns ofspecific gene copy number and/or gene expression within cells andtissues.

Various types of FISH probes can be used to detect chromosometranslocations. Dual color, single fusion probes can be useful indetecting cells possessing a specific chromosomal translocation. The DNAprobe hybridization targets are located on one side of each of the twogenetic breakpoints. “Extra signal” probes can reduce the frequency ofnormal cells exhibiting an abnormal FISH pattern due to the randomco-localization of probe signals in a normal nucleus. One large probespans one breakpoint, while the other probe flanks the breakpoint on theother gene. Dual color, break apart probes are useful in cases wherethere may be multiple translocation partners associated with a knowngenetic breakpoint. This labeling scheme features two differentlycolored probes that hybridize to targets on opposite sides of abreakpoint in one gene. Dual color, dual fusion probes can reduce thenumber of normal nuclei exhibiting abnormal signal patterns. The probeoffers advantages in detecting low levels of nuclei possessing a simplebalanced translocation. Large probes span two breakpoints on differentchromosomes. Such probes are available as Vysis probes from AbbottLaboratories, Abbott Park, Ill.

CISH, or chromogenic in situ hybridization, is a process in which alabeled complementary DNA or RNA strand is used to localize a specificDNA or RNA sequence in a tissue specimen. CISH methodology can be usedto evaluate gene amplification, gene deletion, chromosome translocation,and chromosome number. CISH can use conventional enzymatic detectionmethodology, e.g., horseradish peroxidase or alkaline phosphatasereactions, visualized under a standard bright-field microscope. In acommon embodiment, a probe that recognizes the sequence of interest iscontacted with a sample. An antibody or other binding agent thatrecognizes the probe, e.g., via a label carried by the probe, can beused to target an enzymatic detection system to the site of the probe.In some systems, the antibody can recognize the label of a FISH probe,thereby allowing a sample to be analyzed using both FISH and CISHdetection. CISH can be used to evaluate nucleic acids in multiplesettings, e.g., formalin-fixed, paraffin-embedded (FFPE) tissue, bloodor bone marrow smear, metaphase chromosome spread, and/or fixed cells.In an embodiment, CISH is performed following the methodology in theSPoT-Light® HER2 CISH Kit available from Life Technologies (Carlsbad,Calif.) or similar CISH products available from Life Technologies. TheSPoT-Light® HER2 CISH Kit itself is FDA approved for in vitrodiagnostics and can be used for molecular profiling of HER2. CISH can beused in similar applications as FISH. Thus, one of skill will appreciatethat reference to molecular profiling using FISH herein can be performedusing CISH, unless otherwise specified.

Silver-enhanced in situ hybridization (SISH) is similar to CISH, butwith SISH the signal appears as a black coloration due to silverprecipitation instead of the chromogen precipitates of CISH.

Modifications of the in situ hybridization techniques can be used formolecular profiling according to the invention. Such modificationscomprise simultaneous detection of multiple targets, e.g., Dual ISH,Dual color CISH, bright field double in situ hybridization (BDISH). Seee.g., the FDA approved INFORM HER2 Dual ISH DNA Probe Cocktail kit fromVentana Medical Systems, Inc. (Tucson, Ariz.); DuoCISH™, a dual colorCISH kit developed by Dako Denmark A/S (Denmark).

Comparative Genomic Hybridization (CGH) comprises a molecularcytogenetic method of screening tumor samples for genetic changesshowing characteristic patterns for copy number changes at chromosomaland subchromosomal levels. Alterations in patterns can be classified asDNA gains and losses. CGH employs the kinetics of in situ hybridizationto compare the copy numbers of different DNA or RNA sequences from asample, or the copy numbers of different DNA or RNA sequences in onesample to the copy numbers of the substantially identical sequences inanother sample. In many useful applications of CGH, the DNA or RNA isisolated from a subject cell or cell population. The comparisons can bequalitative or quantitative. Procedures are described that permitdetermination of the absolute copy numbers of DNA sequences throughoutthe genome of a cell or cell population if the absolute copy number isknown or determined for one or several sequences. The differentsequences are discriminated from each other by the different locationsof their binding sites when hybridized to a reference genome, usuallymetaphase chromosomes but in certain cases interphase nuclei. The copynumber information originates from comparisons of the intensities of thehybridization signals among the different locations on the referencegenome. The methods, techniques and applications of CGH are known, suchas described in U.S. Pat. No. 6,335,167, and in U.S. App. Ser. No.60/804,818, the relevant parts of which are herein incorporated byreference.

In an embodiment, CGH used to compare nucleic acids between diseased andhealthy tissues. The method comprises isolating DNA from disease tissues(e.g., tumors) and reference tissues (e.g., healthy tissue) and labelingeach with a different “color” or fluor. The two samples are mixed andhybridized to normal metaphase chromosomes. In the case of array ormatrix CGH, the hybridization mixing is done on a slide with thousandsof DNA probes. A variety of detection system can be used that basicallydetermine the color ratio along the chromosomes to determine DNA regionsthat might be gained or lost in the diseased samples as compared to thereference.

Molecular Profiling for Treatment Selection

The methods of the invention provide a candidate treatment selection fora subject in need thereof. Molecular profiling can be used to identifyone or more candidate therapeutic agents for an individual sufferingfrom a condition in which one or more of the biomarkers disclosed hereinare targets for treatment. For example, the method can identify one ormore chemotherapy treatments for a cancer. In an aspect, the inventionprovides a method comprising: performing at least one molecularprofiling technique on at least one biomarker. Any relevant biomarkercan be assessed using one or more of the molecular profiling techniquesdescribed herein or known in the art. The marker need only have somedirect or indirect association with a treatment to be useful. Anyrelevant molecular profiling technique can be performed, such as thosedisclosed here. These can include without limitation, protein andnucleic acid analysis techniques. Protein analysis techniques include,by way of non-limiting examples, immunoassays, immunohistochemistry, andmass spectrometry. Nucleic acid analysis techniques include, by way ofnon-limiting examples, amplification, polymerase chain amplification,hybridization, microarrays, in situ hybridization, sequencing,dye-terminator sequencing, next generation sequencing, pyrosequencing,and restriction fragment analysis.

Molecular profiling may comprise the profiling of at least one gene (orgene product) for each assay technique that is performed. Differentnumbers of genes can be assayed with different techniques. Any markerdisclosed herein that is associated directly or indirectly with a targettherapeutic can be assessed. For example, any “druggable target”comprising a target that can be modulated with a therapeutic agent suchas a small molecule or binding agent such as an antibody, is a candidatefor inclusion in the molecular profiling methods of the invention. Thetarget can also be indirectly drug associated, such as a component of abiological pathway that is affected by the associated drug. Themolecular profiling can be based on either the gene, e.g., DNA sequence,and/or gene product, e.g., mRNA or protein. Such nucleic acid and/orpolypeptide can be profiled as applicable as to presence or absence,level or amount, activity, mutation, sequence, haplotype, rearrangement,copy number, or other measurable characteristic. In some embodiments, asingle gene and/or one or more corresponding gene products is assayed bymore than one molecular profiling technique. A gene or gene product(also referred to herein as “marker” or “biomarker”), e.g., an mRNA orprotein, is assessed using applicable techniques (e.g., to assess DNA,RNA, protein), including without limitation ISH, gene expression, IHC,sequencing or immunoassay. Therefore, any of the markers disclosedherein can be assayed by a single molecular profiling technique or bymultiple methods disclosed herein (e.g., a single marker is profiled byone or more of IHC, ISH, sequencing, microarray, etc.). In someembodiments, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or at least about 100genes or gene products are profiled by at least one technique, aplurality of techniques, or using a combination of ISH, gene expression,gene copy, IHC, and sequencing. In some embodiments, at least about 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000,16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000,25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000,34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000,43,000, 44,000, 45,000, 46,000, 47,000, 48,000, 49,000, or at least50,000 genes or gene products are profiled using various techniques. Thenumber of markers assayed can depend on the technique used. For example,microarray and massively parallel sequencing lend themselves to highthroughput analysis. Because molecular profiling queries molecularcharacteristics of the tumor itself, this approach provides informationon therapies that might not otherwise be considered based on the lineageof the tumor.

In some embodiments, a sample from a subject in need thereof is profiledusing methods which include but are not limited to IHC analysis, geneexpression analysis, ISH analysis, and/or sequencing analysis (such asby PCR, RT-PCR, pyrosequencing) for one or more of the following: ABCC1,ABCG2, ACE2, ADA, ADH1C, ADH4, AGT, AR, AREG, ASNS, BCL2, BCRP, BDCA1,beta III tubulin, BIRC5, B-RAF, BRCA1, BRCA2, CA2, caveolin, CD20, CD25,CD33, CD52, CDA, CDKN2A, CDKN1A, CDKN1B, CDK2, CDW52, CES2, CK 14, CK17, CK 5/6, c-KIT, c-Met, c-Myc, COX-2, Cyclin D1, DCK, DHFR, DNMT1,DNMT3A, DNMT3B, E-Cadherin, ECGF1, EGFR, EML4-ALK fusion, EPHA2,Epiregulin, ER, ERBR2, ERCC1, ERCC3, EREG, ESR1, FLT1, folate receptor,FOLR1, FOLR2, FSHB, FSHPRH1, FSHR, FYN, GART, GNA11, GNAQ, GNRH1,GNRHR1, GSTP1, HCK, HDAC1, hENT-1, Her2/Neu, HGF, HIF1A, HIG1, HSP90,HSP9OAA1, HSPCA, IGF-1R, IGFRBP, IGFRBP3, IGFRBP4, IGFRBP5, IL13RA1,IL2RA, KDR, Ki67, KIT, K-RAS, LCK, LTB, Lymphotoxin Beta Receptor, LYN,MET, MGMT, MLH1, MMR, MRP1, MS4A1, MSH2, MSHS, Myc, NFKB1, NFKB2,NFKBIA, NRAS, ODC1, OGFR, p16, p21, p27, p53, p95, PARP-1, PDGFC, PDGFR,PDGFRA, PDGFRB, PGP, PGR, PI3K, POLA, POLA1, PPARG, PPARGC1, PR, PTEN,PTGS2, PTPN12, RAF1, RARA, ROS1, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2,SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, Survivin, TK1, TLE3, TNF,TOP1, TOP2A, TOP2B, TS, TUBB3, TXN, TXNRD1, TYMS, VDR, VEGF, VEGFA,VEGFC, VHL, YES1, ZAP70.

Table 2 provides a listing of gene and corresponding protein symbols andnames of many of the molecular profiling targets that are analyzedaccording to the methods of the invention. As understood by those ofskill in the art, genes and proteins have developed a number ofalternative names in the scientific literature. Thus, the listing inTable 2 comprises an illustrative but not exhaustive compilation. Afurther listing of gene aliases and descriptions can be found using avariety of online databases, including GeneCards® (www.genecards.org),HUGO Gene Nomenclature (www.genenames.org), Entrez Gene(www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene), UniProtKB/Swiss-Prot(www.uniprot.org), UniProtKB/TrEMBL (www.uniprot.org), OMIM(www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM), GeneLoc(genecards.weizmann.ac.il/geneloc/), and Ensembl (www.ensembl.org).Generally, gene symbols and names below correspond to those approved byHUGO, and protein names are those recommended by UniProtKB/Swiss-Prot.Common alternatives are provided as well. Where a protein name indicatesa precursor, the mature protein is also implied. Throughout theapplication, gene and protein symbols may be used interchangeably andthe meaning can be derived from context, e.g., FISH is used to analyzenucleic acids whereas IHC is used to analyze protein.

TABLE 2 Gene and Protein Names Gene Protein Symbol Gene Name SymbolProtein Name ABCB1, ATP-binding cassette, sub-family B ABCB1, Multidrugresistance protein 1; P- PGP (MDR/TAP), member 1 MDR1, PGP glycoproteinABCC1, ATP-binding cassette, sub-family C MRP 1, Multidrugresistance-associated protein MRP1 (CFTR/MRP), member 1 ABCC1 1 ABCG2,ATP-binding cassette, sub-family G ABCG2 ATP-binding cassette sub-familyG BCRP (WHITE), member 2 member 2 ACE2 angiotensin I converting enzymeACE2 Angiotensin-converting enzyme 2 (peptidyl-dipeptidase A) 2precursor ADA adenosine deaminase ADA Adenosine deaminase ADH1C alcoholdehydrogenase 1C (class I), ADH1G Alcohol dehydrogenase 1C gammapolypeptide ADH4 alcohol dehydrogenase 4 (class II), pi ADH4 Alcoholdehydrogenase 4 polypeptide AGT angiotensinogen (serpin peptidase ANGT,AGT Angiotensinogen precursor inhibitor, clade A, member 8) ALKanaplastic lymphoma receptor ALK ALK tyrosine kinase receptor precursortyrosine kinase AR androgen receptor AR Androgen receptor AREGamphiregulin AREG Amphiregulin precursor ASNS asparagine synthetase ASNSAsparagine synthetase [glutamine- hydrolyzing] BCL2 B-cell CLL/lymphoma2 BCL2 Apoptosis regulator Bcl-2 BDCA1, CD1c molecule CD1C T-cellsurface glycoprotein CD1c CD1C precursor BIRC5 baculoviral IAPrepeat-containing 5 BIRC5, Baculoviral IAP repeat-containing Survivinprotein 5; Survivin BRAF v-raf murine sarcoma viral oncogene B-RAF,Serine/threonine-protein kinase B-raf homolog B1 BRAF BRCA1 breastcancer 1, early onset BRCA1 Breast cancer type 1 susceptibility proteinBRCA2 breast cancer 2, early onset BRCA2 Breast cancer type 2susceptibility protein CA2 carbonic anhydrase II CA2 Carbonic anhydrase2 CAV1 caveolin 1, caveolae protein, 22 kDa CAV1 Caveolin-1 CCND1 cyclinD1 CCND1, G1/S-specific cyclin-D1 Cyclin D1, BCL-1 CD20,membrane-spanning 4-domains, CD20 B-lymphocyte antigen CD20 MS4A1subfamily A, member 1 CD25, interleukin 2 receptor, alpha CD25Interleukin-2 receptor subunit alpha IL2RA precursor CD33 CD33 moleculeCD33 Myeloid cell surface antigen CD33 precursor CD52, CD52 moleculeCD52 CAMPATH-1 antigen precursor CDW52 CDA cytidine deaminase CDACytidine deaminase CDH1, cadherin 1, type 1, E-cadherin E-Cad Cadherin-1precursor (E-cadherin) ECAD (epithelial) CDK2 cyclin-dependent kinase 2CDK2 Cell division protein kinase 2 CDKN1A, cyclin-dependent kinaseinhibitor 1A CDKN1A, Cyclin-dependent kinase inhibitor 1 P21 (p21, Cip1)p21 CDKN1B cyclin-dependent kinase inhibitor 1B CDKN1B, Cyclin-dependentkinase inhibitor 1B (p27, Kip1) p27 CDKN2A, cyclin-dependent kinaseinhibitor 2A CD21A, p16 Cyclin-dependent kinase inhibitor 2A, P16(melanoma, p16, inhibits CDK4) isoforms 1/2/3 CES2 carboxylesterase 2(intestine, liver) CES2, EST2 Carboxylesterase 2 precursor CK 5/6cytokeratin 5/cytokeratin 6 CK 5/6 Keratin, type II cytoskeletal 5;Keratin, type II cytoskeletal 6 CK14, keratin 14 CK14 Keratin, type Icytoskeletal 14 KRT14 CK17, keratin 17 CK17 Keratin, type I cytoskeletal17 KRT17 COX2, prostaglandin-endoperoxide synthase COX-2, ProstaglandinG/H synthase 2 precursor PTGS2 2 (prostaglandin G/H synthase and PTGS2cyclooxygenase) DCK deoxycytidine kinase DCK Deoxycytidine kinase DHFRdihydrofolate reductase DHFR Dihydrofolate reductase DNMT1 DNA(cytosine-5-)-methyltransferase DNMT1 DNA (cytosine-5)-methyltransferase1 1 DNMT3A DNA (cytosine-5-)-methyltransferase DNMT3A DNA(cytosine-5)-methyltransferase 3A 3 alpha DNMT3B DNA(cytosine-5-)-methyltransferase DNMT3B DNA(cytosine-5)-methyltransferase 3B 3 beta ECGF1, thymidine phosphorylaseTYMP, PD- Thymidine phosphorylase precursor TYMP ECGF, ECDF1 EGFR,epidermal growth factor receptor EGFR, Epidermal growth factor receptorERBB1, (erythroblastic leukemia viral (v-erb- ERBB1, precursor HER1 b)oncogene homolog, avian) HER1 EML4 echinoderm microtubule associatedEML4 Echinoderm microtubule-associated protein like 4 protein-like 4EPHA2 EPH receptor A2 EPHA2 Ephrin type-A receptor 2 precursor ER, ESR1estrogen receptor 1 ER, ESR1 Estrogen receptor ERBB2, v-erb-b2erythroblastic leukemia ERBB2, Receptor tyrosine-protein kinase erbB-2HER2/NEU viral oncogene homolog 2, HER2, HER- precursorneuro/glioblastoma derived oncogene 2/neu homolog (avian) ERCC1 excisionrepair cross-complementing ERCC1 DNA excision repair protein ERCC-1rodent repair deficiency, complementation group 1 (includes overlappingantisense sequence) ERCC3 excision repair cross-complementing ERCC3TFIIH basal transcription factor complex rodent repair deficiency,helicase XPB subunit complementation group 3 (xeroderma pigmentosumgroup B complementing) EREG Epiregulin EREG Proepiregulin precursor FLT1fms-related tyrosine kinase 1 FLT-1, Vascular endothelial growth factor(vascular endothelial growth VEGFR1 receptor 1 precursor factor/vascularpermeability factor receptor) FOLR1 folate receptor 1 (adult) FOLR1Folate receptor alpha precursor FOLR2 folate receptor 2 (fetal) FOLR2Folate receptor beta precursor FSHB follicle stimulating hormone, betaFSHB Follitropin subunit beta precursor polypeptide FSHPRH1, centromereprotein I FSHPRH1, Centromere protein I CENP1 CENP1 FSHR folliclestimulating hormone FSHR Follicle-stimulating hormone receptor receptorprecursor FYN FYN oncogene related to SRC, FGR, FYN Tyrosine-proteinkinase Fyn YES GART phosphoribosylglycinamide GART, Trifunctional purinebiosynthetic protein formyltransferase, PUR2 adenosine-3phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazolesynthetase GNA11, guanine nucleotide binding protein GNA11, G Guaninenucleotide-binding protein GA11 (G protein), alpha 11 (Gq class)alpha-11, G- subunit alpha-11 protein subunit alpha-11 GNAQ, guaninenucleotide binding protein GNAQ Guanine nucleotide-binding protein G(q)GAQ (G protein), q polypeptide subunit alpha GNRH1gonadotropin-releasing hormone 1 GNRH1, Progonadoliberin-1 precursor(luteinizing-releasing hormone) GON1 GNRHR1, gonadotropin-releasinghormone GNRHR1 Gonadotropin-releasing hormone GNRHR receptor receptorGSTP1 glutathione S-transferase pi 1 GSTP1 Glutathione S-transferase PHCK hemopoietic cell kinase HCK Tyrosine-protein kinase HCK HDAC1histone deacetylase 1 HDAC1 Histone deacetylase 1 HGF hepatocyte growthfactor HGF Hepatocyte growth factor precursor (hepapoietin A; scatterfactor) HIF1A hypoxia inducible factor 1, alpha HIF1A Hypoxia-induciblefactor 1-alpha subunit (basic helix-loop-helix transcription factor)HIG1, HIG1 hypoxia inducible domain HIG1, HIG1 domain family member 1AHIGD1A, family, member 1A HIGD1A, HIG1A HIG1A HSP90AA1, heat shockprotein 90 kDa alpha HSP90, Heat shock protein HSP 90-alpha HSP90,(cytosolic), class A member 1 HSP90A HSPCA IGF1R insulin-like growthfactor 1 receptor IGF-1R Insulin-like growth factor 1 receptor precursorIGFBP3, insulin-like growth factor binding IGFBP-3, Insulin-like growthfactor-binding IGFRBP3 protein 3 IBP-3 protein 3 precursor IGFBP4,insulin-like growth factor binding IGFBP-4, Insulin-like growthfactor-binding IGFRBP4 protein 4 IBP-4 protein 4 precursor IGFBP5,insulin-like growth factor binding IGFBP-5, Insulin-like growthfactor-binding IGFRBP5 protein 5 IBP-5 protein 5 precursor IL13RA1interleukin 13 receptor, alpha 1 IL-13RA1 Interleukin-13 receptorsubunit alpha-1 precursor KDR kinase insert domain receptor (a type KDR,Vascular endothelial growth factor III receptor tyrosine kinase) VEGFR2receptor 2 precursor KIT, c-KIT v-kit Hardy-Zuckerman 4 feline KIT,c-KIT, Mast/stem cell growth factor receptor sarcoma viral oncogenehomolog CD117, precursor SCFR KRAS v-Ki-ras2 Kirsten rat sarcoma viralK-RAS GTPase KRas precursor oncogene homolog LCK lymphocyte-specificprotein tyrosine LCK Tyrosine-protein kinase Lck kinase LTB lymphotoxinbeta (TNF superfamily, LTB, TNF3 Lymphotoxin-beta member 3) LTBRlymphotoxin beta receptor (TNFR LTBR, Tumor necrosis factor receptorsuperfamily, member 3) LTBR3, superfamily member 3 precursor TNFR LYNv-yes-1 Yamaguchi sarcoma viral LYN Tyrosine-protein kinase Lyn relatedoncogene homolog MET, c- met proto-oncogene (hepatocyte MET, c-Hepatocyte growth factor receptor MET growth factor receptor) METprecursor MGMT O-6-methylguanine-DNA MGMTMethylated-DNA--protein-cysteine methyltransferase methyltransferaseMKI67, antigen identified by monoclonal Ki67, Ki-67 Antigen KI-67 KI67antibody Ki-67 MLH1 mutL homolog 1, colon cancer, MLH1 DNA mismatchrepair protein Mlhl nonpolyposis type 2 (E. coli) MMR mismatch repair(refers to MLH1, MSH2, MSH5) MSH2 mutS homolog 2, colon cancer, MSH2 DNAmismatch repair protein Msh2 nonpolyposis type 1 (E. coli) MSH5 mutShomolog 5 (E. coli) MSH5, MutS protein homolog 5 hMSH5 MYC, c- v-mycmyelocytomatosis viral MYC, c- Myc proto-oncogene protein MYC oncogenehomolog (avian) MYC NBN, P95 nibrin NBN, p95 Nibrin NDGR1 N-mycdownstream regulated 1 NDGR1 Protein NDGR1 NFKB1 nuclear factor of kappalight NFKB1 Nuclear factor NF-kappa-B p105 polypeptide gene enhancer inB-cells subunit 1 NFKB2 nuclear factor of kappa light NFKB2 Nuclearfactor NF-kappa-B p100 subunit polypeptide gene enhancer in B-cells 2(p49/p100) NFKBIA nuclear factor of kappa light NFKBIA NF-kappa-Binhibitor alpha polypeptide gene enhancer in B-cells inhibitor, alphaNRAS neuroblastoma RAS viral (v-ras) NRAS GTPase NRas, Transformingprotein N- oncogene homolog Ras ODC1 ornithine decarboxylase 1 ODCOrnithine decarboxylase OGFR opioid growth factor receptor OGFR Opioidgrowth factor receptor PARP1 poly (ADP-ribose) polymerase 1 PARP-1 Poly[ADP-ribose] polymerase 1 PDGFC platelet derived growth factor C PDGF-C,Platelet-derived growth factor C VEGF-E precursor PDGFR platelet-derivedgrowth factor PDGFR Platelet-derived growth factor receptor receptorPDGFRA platelet-derived growth factor PDGFRA, Alpha-typeplatelet-derived growth receptor, alpha polypeptide PDGFR2, factorreceptor precursor CD140 A PDGFRB platelet-derived growth factor PDGFRB,Beta-type platelet-derived growth factor receptor, beta polypeptidePDGFR, receptor precursor PDGFR1, CD140 B PGR progesterone receptor PRProgesterone receptor PIK3CA phosphoinositide-3-kinase, catalytic, PI3Ksubunit phosphoinositide-3-kinase, catalytic, alpha polypeptide p110αalpha polypeptide POLA1 polymerase (DNA directed), alpha 1, POLA, DNApolymerase alpha catalytic subunit catalytic subunit; polymerase (DNAPOLA1, directed), alpha, polymerase (DNA p180 directed), alpha 1 PPARG,peroxisome proliferator-activated PPARG Peroxisomeproliferator-activated PPARG1, receptor gamma receptor gamma PPARG2,PPAR- gamma, NR1C3 PPARGC1A, peroxisome proliferator-activated PGC-1-Peroxisome proliferator-activated LEM6, receptor gamma, coactivator 1alpha alpha, receptor gamma coactivator 1-alpha; PGC1, PPARGC-1-PPAR-gamma coactivator 1-alpha PGC1A, alpha PPARGC1 PSMD9, proteasome(prosome, macropain) p27 26S proteasome non-ATPase regulatory P27 26Ssubunit, non-ATPase, 9 subunit 9 PTEN, phosphatase and tensin homologPTEN Phosphatidylinositol-3,4,5-trisphosphate MMAC1, 3-phosphatase anddual-specificity TEP1 protein phosphatase; Mutated in multiple advancedcancers 1 PTPN12 protein tyrosine phosphatase, non- PTPG1Tyrosine-protein phosphatase non- receptor type 12 receptor type 12;Protein-tyrosine phosphatase G1 RAF1 v-raf-1 murine leukemia viral RAF,RAF- RAF proto-oncogene serine/threonine- oncogene homolog 1 1, c-RAFprotein kinase RARA retinoic acid receptor, alpha RAR, RAR- Retinoicacid receptor alpha alpha, RARA ROS1, c-ros oncogene 1, receptortyrosine ROS1, ROS Proto-oncogene tyrosine-protein kinase ROS, kinaseROS MCF3 RRM1 ribonucleotide reductase M1 RRM1, RR1Ribonucleoside-diphosphate reductase large subunit RRM2 ribonucleotidereductase M2 RRM2, Ribonucleoside-diphosphate reductase RR2M, RR2subunit M2 RRM2B ribonucleotide reductase M2 B (TP53 RRM2B,Ribonucleoside-diphosphate reductase inducible) P53R2 subunit M2 B RXRBretinoid X receptor, beta RXRB Retinoic acid receptor RXR-beta RXRGretinoid X receptor, gamma RXRG, Retinoic acid receptor RXR-gamma RXRCSIK2 salt-inducible kinase 2 SIK2, Salt-inducible protein kinase 2;Q9H0K1 Serine/threonine-protein kinase SIK2 SLC29A1 solute carrierfamily 29 (nucleoside ENT-1 Equilibrative nucleoside transporter 1transporters), member 1 SPARC secreted protein, acidic, cysteine-richSPARC SPARC precursor; Osteonectin (osteonectin) SRC v-src sarcoma(Schmidt-Ruppin A-2) SRC Proto-oncogene tyrosine-protein kinase viraloncogene homolog (avian) Src SSTR1 somatostatin receptor 1 SSTR1,Somatostatin receptor type 1 SSR1, SS1R SSTR2 somatostatin receptor 2SSTR2, Somatostatin receptor type 2 SSR2, SS2R SSTR3 somatostatinreceptor 3 SSTR3, Somatostatin receptor type 3 SSR3, SS3R SSTR4somatostatin receptor 4 SSTR4, Somatostatin receptor type 4 SSR4, SS4RSSTR5 somatostatin receptor 5 SSTR5, Somatostatin receptor type 5 SSR5,SS5R TK1 thymidine kinase 1, soluble TK1, KITH Thymidine kinase,cytosolic TLE3 transducin-like enhancer of split 3 TLE3 Transducin-likeenhancer protein 3 (E(sp1) homolog, Drosophila) TNF tumor necrosisfactor (TNF TNF, TNF- Tumor necrosis factor precursor superfamily,member 2) alpha, TNF-a TOP1, topoisomerase (DNA) I TOP1, DNAtopoisomerase 1 TOPO1 TOPO1 TOP2A, topoisomerase (DNA) II alpha TOP2A,DNA topoisomerase 2-alpha; TOPO2A 170 kDa TOP2, Topoisomerase II alphaTOPO2A TOP2B, topoisomerase (DNA) II beta TOP2B, DNA topoisomerase2-beta; TOPO2B 180 kDa TOPO2B Topoisomerase II beta TP53 tumor proteinp53 p53 Cellular tumor antigen p53 TUBB3 tubulin, beta 3 Beta IIITubulin beta-3 chain tubulin, TUBB3, TUBB4 TXN thioredoxin TXN, TRX,Thioredoxin TRX-1 TXNRD1 thioredoxin reductase 1 TXNRD1, Thioredoxinreductase 1, cytoplasmic; TXNR Oxidoreductase TYMS, TS thymidylatesynthetase TYMS, TS Thymidylate synthase VDR vitamin D(1,25-dihydroxyvitamin VDR Vitamin D3 receptor D3) receptor VEGFA,vascular endothelial growth factor A VEGF-A, Vascular endothelial growthfactor A VEGF VEGF precursor VEGFC vascular endothelial growth factor CVEGF-C Vascular endothelial growth factor C precursor VHL vonHippel-Lindau tumor suppressor VHL Von Hippel-Lindau disease tumorsuppressor YES1 v-yes-1 Yamaguchi sarcoma viral YES1, Yes,Proto-oncogene tyrosine-protein kinase oncogene homolog 1 p61-Yes YesZAP70 zeta-chain (TCR) associated protein ZAP-70 Tyrosine-protein kinaseZAP-70 kinase 70 kDa

In some embodiments, additional molecular profiling methods areperformed. These can include without limitation PCR, RT-PCR, Q-PCR,SAGE, MPSS, immunoassays and other techniques to assess biologicalsystems described herein or known to those of skill in the art. Thechoice of genes and gene products to be assayed can be updated over timeas new treatments and new drug targets are identified. Once theexpression or mutation of a biomarker is correlated with a treatmentoption, it can be assessed by molecular profiling. One of skill willappreciate that such molecular profiling is not limited to thosetechniques disclosed herein but comprises any methodology conventionalfor assessing nucleic acid or protein levels, sequence information, orboth. The methods of the invention can also take advantage of anyimprovements to current methods or new molecular profiling techniquesdeveloped in the future. In some embodiments, a gene or gene product isassessed by a single molecular profiling technique. In otherembodiments, a gene and/or gene product is assessed by multiplemolecular profiling techniques. In a non-limiting example, a genesequence can be assayed by one or more of FISH and pyrosequencinganalysis, the mRNA gene product can be assayed by one or more of RT-PCRand microarray, and the protein gene product can be assayed by one ormore of IHC and immunoassay. One of skill will appreciate that anycombination of biomarkers and molecular profiling techniques that willbenefit disease treatment are contemplated by the invention.

Genes and gene products that are known to play a role in cancer and canbe assayed by any of the molecular profiling techniques of the inventioninclude without limitation 2AR, A DISINTEGRIN, ACTIVATOR OF THYROID ANDRETINOIC ACID RECEPTOR (ACTR), ADAM 11, ADIPOGENESIS INHIBITORY FACTOR(ADIF), ALPHA 6 INTEGRIN SUBUNIT, ALPHA V INTEGRIN SUBUNIT,ALPHA-CATENIN, AMPLIFIED IN BREAST CANCER 1 (AIB1), AMPLIFIED IN BREASTCANCER 3 (AIB3), AMPLIFIED IN BREAST CANCER 4 (AIB4), AMYLOID PRECURSORPROTEIN SECRETASE (APPS), AP-2 GAMMA, APPS, ATP-BINDING CASSETTETRANSPORTER (ABCT), PLACENTA-SPECIFIC (ABCP), ATP-BINDING CASSETTESUBFAMILY C MEMBER (ABCC1), BAG-1, BASIGIN (BSG), BCEI, B-CELLDIFFERENTIATION FACTOR (BCDF), B-CELL LEUKEMIA 2 (BCL-2), B-CELLSTIMULATORY FACTOR-2 (BSF-2), BCL-1, BCL-2-ASSOCIATED X PROTEIN (BAX),BCRP, BETA 1 INTEGRIN SUBUNIT, BETA 3 INTEGRIN SUBUNIT, BETA 5 INTEGRINSUBUNIT, BETA-2 INTERFERON, BETA-CATENIN, BETA-CATENIN, BONESIALOPROTEIN (BSP), BREAST CANCER ESTROGEN-INDUCIBLE SEQUENCE (BCEI),BREAST CANCER RESISTANCE PROTEIN (BCRP), BREAST CANCER TYPE 1 (BRCA1),BREAST CANCER TYPE 2 (BRCA2), BREAST CARCINOMA AMPLIFIED SEQUENCE 2(BCAS2), CADHERIN, EPITHELIAL CADHERIN-11, CADHERIN-ASSOCIATED PROTEIN,CALCITONIN RECEPTOR (CTR), CALCIUM PLACENTAL PROTEIN (CAPL), CALCYCLIN,CALLA, CAMS, CAPL, CARCINOEMBRYONIC ANTIGEN (CEA), CATENIN, ALPHA 1,CATHEPSIN B, CATHEPSIN D, CATHEPSIN K, CATHEPSIN L2, CATHEPSIN 0,CATHEPSIN 01, CATHEPSIN V, CD10, CD146, CD147, CD24, CD29, CD44, CD51,CD54, CD61, CD66e, CD82, CD87, CD9, CEA, CELLULAR RETINOL-BINDINGPROTEIN 1 (CRBP1), c-ERBB-2, CK7, CK8, CK18, CK19, CK20, CLAUDIN-7,c-MET, COLLAGENASE, FIBROBLAST, COLLAGENASE, INTERSTITIAL,COLLAGENASE-3, COMMON ACUTE LYMPHOCYTIC LEUKEMIA ANTIGEN (CALLA),CONNEXIN 26 (Cx26), CONNEXIN 43 (Cx43), CORTACTIN, COX-2, CTLA-8, CTR,CTSD, CYCLIN D1, CYCLOOXYGENASE-2, CYTOKERATIN 18, CYTOKERATIN 19,CYTOKERATIN 8, CYTOTOXIC T-LYMPHOCYTE-ASSOCIATED SERINE ESTERASE 8(CTLA-8), DIFFERENTIATION-INHIBITING ACTIVITY (DIA), DNA AMPLIFIED INMAMMARY CARCINOMA 1 (DAM1), DNA TOPOISOMERASE II ALPHA, DR-NM23,E-CADHERIN, EMMPRIN, EMS1, ENDOTHELIAL CELL GROWTH FACTOR (ECGR),PLATELET-DERIVED (PD-ECGF), ENKEPHALINASE, EPIDERMAL GROWTH FACTORRECEPTOR (EGFR), EPISIALIN, EPITHELIAL MEMBRANE ANTIGEN (EMA), ER-ALPHA,ERBB2, ERBB4, ER-BETA, ERF-1, ERYTHROID-POTENTIATING ACTIVITY (EPA),ESR1, ESTROGEN RECEPTOR-ALPHA, ESTROGEN RECEPTOR-BETA, ETS-1,EXTRACELLULAR MATRIX METALLOPROTEINASE INDUCER (EMMPRIN), FIBRONECTINRECEPTOR, BETA POLYPEPTIDE (FNRB), FIBRONECTIN RECEPTOR BETA SUBUNIT(FNRB), FLK-1, GA15.3, GA733.2, GALECTIN-3, GAMMA-CATENIN, GAP JUNCTIONPROTEIN (26 kDa), GAP JUNCTION PROTEIN (43 kDa), GAP JUNCTION PROTEINALPHA-1 (GJA1), GAP JUNCTION PROTEIN BETA-2 (GJB2), GCP1, GELATINASE A,GELATINASE B, GELATINASE (72 kDa), GELATINASE (92 kDa), GLIOSTATIN,GLUCOCORTICOID RECEPTOR INTERACTING PROTEIN 1 (GRIP1), GLUTATHIONES-TRANSFERASE p, GM-CSF, GRANULOCYTE CHEMOTACTIC PROTEIN 1 (GCP1),GRANULOCYTE-MACROPHAGE-COLONY STIMULATING FACTOR, GROWTH FACTOR RECEPTORBOUND-7 (GRB-7), GSTp, HAP, HEAT-SHOCK COGNATE PROTEIN 70 (HSC70),HEAT-STABLE ANTIGEN, HEPATOCYTE GROWTH FACTOR (HGF), HEPATOCYTE GROWTHFACTOR RECEPTOR (HGFR), HEPATOCYTE-STIMULATING FACTOR III (HSF III),HER-2, HER2/NEU, HERMES ANTIGEN, HET, HHM, HUMORAL HYPERCALCEMIA OFMALIGNANCY (HHM), ICERE-1, INT-1, INTERCELLULAR ADHESION MOLECULE-1(ICAM-1), INTERFERON-GAMMA-INDUCING FACTOR (IGIF), INTERLEUKIN-1 ALPHA(IL-1A), INTERLEUKIN-1 BETA (IL-1B), INTERLEUKIN-11 (IL-11),INTERLEUKIN-17 (IL-17), INTERLEUKIN-18 (IL-18), INTERLEUKIN-6 (IL-6),INTERLEUKIN-8 (IL-8), INVERSELY CORRELATED WITH ESTROGEN RECEPTOREXPRESSION-1 (ICERE-1), KAI1, KDR, KERATIN 8, KERATIN 18, KERATIN 19,KISS-1, LEUKEMIA INHIBITORY FACTOR (LIF), LIF, LOST IN INFLAMMATORYBREAST CANCER (LIBC), LOT (“LOST ON TRANSFORMATION”), LYMPHOCYTE HOMINGRECEPTOR, MACROPHAGE-COLONY STIMULATING FACTOR, MAGE-3, MAMMAGLOBIN,MASPIN, MC56, M-CSF, MDC, MDNCF, MDR, MELANOMA CELL ADHESION MOLECULE(MCAM), MEMBRANE METALLOENDOPEPTIDASE (MME), MEMBRANE-ASSOCIATED NEUTRALENDOPEPTIDASE (NEP), CYSTEINE-RICH PROTEIN (MDC), METASTASIN (MTS-1),MLN64, MMP1, MMP2, MMP3, MMP1, MMP9, MMP11, MMP13, MMP14, MMP15, MMP16,MMP17, MOESIN, MONOCYTE ARGININE-SERPIN, MONOCYTE-DERIVED NEUTROPHILCHEMOTACTIC FACTOR, MONOCYTE-DERIVED PLASMINOGEN ACTIVATOR INHIBITOR,MTS-1, MUC-1, MUC18, MUCIN LIKE CANCER ASSOCIATED ANTIGEN (MCA), MUCIN,MUC-1, MULTIDRUG RESISTANCE PROTEIN 1 (MDR, MDR1), MULTIDRUG RESISTANCERELATED PROTEIN-1 (MRP, MRP-1), N-CADHERIN, NEP, NEU, NEUTRALENDOPEPTIDASE, NEUTROPHIL-ACTIVATING PEPTIDE 1 (NAP1), NM23-H1, NM23-H2,NME1, NME2, NUCLEAR RECEPTOR COACTIVATOR-1 (NCoA-1), NUCLEAR RECEPTORCOACTIVATOR-2 (NCoA-2), NUCLEAR RECEPTOR COACTIVATOR-3 (NCoA-3),NUCLEOSIDE DIPHOSPHATE KINASE A (NDPKA), NUCLEOSIDE DIPHOSPHATE KINASE B(NDPKB), ONCOSTATIN M (OSM), ORNITHINE DECARBOXYLASE (ODC), OSTEOCLASTDIFFERENTIATION FACTOR (ODF), OSTEOCLAST DIFFERENTIATION FACTOR RECEPTOR(ODFR), OSTEONECTIN (OSN, ON), OSTEOPONTIN (OPN), OXYTOCIN RECEPTOR(OXTR), p27/kipl, p300/CBP COINTEGRATOR ASSOCIATE PROTEIN (p/CIP), p53,p9Ka, PAI-1, PAI-2, PARATHYROID ADENOMATOSIS 1 (PRAD1), PARATHYROIDHORMONE-LIKE HORMONE (PTHLH), PARATHYROID HORMONE-RELATED PEPTIDE(PTHrP), P-CADHERIN, PD-ECGF, PDGF, PEANUT-REACTIVE URINARY MUCIN (PUM),P-GLYCOPROTEIN (P-GP), PGP-1, PHGS-2, PHS-2, PIP, PLAKOGLOBIN,PLASMINOGEN ACTIVATOR INHIBITOR (TYPE 1), PLASMINOGEN ACTIVATORINHIBITOR (TYPE 2), PLASMINOGEN ACTIVATOR (TISSUE-TYPE), PLASMINOGENACTIVATOR (UROKINASE-TYPE), PLATELET GLYCOPROTEIN Ma (GP3A), PLAU,PLEOMORPHIC ADENOMA GENE-LIKE 1 (PLAGL1), POLYMORPHIC EPITHELIAL MUCIN(PEM), PRAD1, PROGESTERONE RECEPTOR (PgR), PROGESTERONE RESISTANCE,PROSTAGLANDIN ENDOPEROXIDE SYNTHASE-2, PROSTAGLANDIN G/H SYNTHASE-2,PROSTAGLANDIN H SYNTHASE-2, pS2, PS6K, PSORIASIN, PTHLH, PTHrP, RAD51,RAD52, RAD54, RAP46, RECEPTOR-ASSOCIATED COACTIVATOR 3 (RAC3), REPRESSOROF ESTROGEN RECEPTOR ACTIVITY (REA), S100A4, S100A6, S100A7, S6K,SART-1, SCAFFOLD ATTACHMENT FACTOR B (SAF-B), SCATTER FACTOR (SF),SECRETED PHOSPHOPROTEIN-1 (SPP-1), SECRETED PROTEIN, ACIDIC AND RICH INCYSTEINE (SPARC), STANNICALCIN, STEROID RECEPTOR COACTIVATOR-1 (SRC-1),STEROID RECEPTOR COACTIVATOR-2 (SRC-2), STEROID RECEPTOR COACTIVATOR-3(SRC-3), STEROID RECEPTOR RNA ACTIVATOR (SRA), STROMELYSIN-1,STROMELYSIN-3, TENASCIN-C (TN-C), TESTES-SPECIFIC PROTEASE 50,THROMBOSPONDIN I, THROMBOSPONDIN II, THYMIDINE PHOSPHORYLASE (TP),THYROID HORMONE RECEPTOR ACTIVATOR MOLECULE 1 (TRAM-1), TIGHT JUNCTIONPROTEIN 1 (TJP1), TIMP1, TIMP2, TIMP3, TIMP4, TISSUE-TYPE PLASMINOGENACTIVATOR, TN-C, TP53, tPA, TRANSCRIPTIONAL INTERMEDIARY FACTOR 2(TIF2), TREFOIL FACTOR 1 (TFF1), TSG101, TSP-1, TSP1, TSP-2, TSP2,TSP50, TUMOR CELL COLLAGENASE STIMULATING FACTOR (TCSF),TUMOR-ASSOCIATED EPITHELIAL MUCIN, uPA, uPAR, UROKINASE, UROKINASE-TYPEPLASMINOGEN ACTIVATOR, UROKINASE-TYPE PLASMINOGEN ACTIVATOR RECEPTOR(uPAR), UVOMORULIN, VASCULAR ENDOTHELIAL GROWTH FACTOR, VASCULARENDOTHELIAL GROWTH FACTOR RECEPTOR-2 (VEGFR2), VASCULAR ENDOTHELIALGROWTH FACTOR-A, VASCULAR PERMEABILITY FACTOR, VEGFR2, VERY LATE T-CELLANTIGEN BETA (VLA-BETA), VIMENTIN, VITRONECTIN RECEPTOR ALPHAPOLYPEPTIDE (VNRA), VITRONECTIN RECEPTOR, VON WILLEBRAND FACTOR, VPF,VWF, WNT-1, ZAC, ZO-1, and ZONULA OCCLUDENS-1.

In some embodiments, IHC is used to detect on or more of the followingproteins, including without limitation: ADA, AR, ASNA, BCL2, BRCA2,c-Met, CD33, CDW52, CES2, DNMT1, EGFR, EML4-ALK fusion, ERBB2, ERCC3,ESR1, FOLR2, GART, GSTP1, HDAC1, hENT-1, HIF1A, HSPCA, IGF-1R, IL2RA,KIT, MLH1, MMR, MS4A1, MASH2, NFKB2, NFKBIA, OGFR, p16, p21, p27,PARP-1, PI3K, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA,RXRB, SPARC, SSTR1, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS,VDR, VEGF, VHL, or ZAP70. The proteins can be detected by IHC usingmonoclonal or polyclonal antibodies. In some embodiments, both are used.As an illustrative example, SPARC can be detected by anti-SPARCmonoclonal (SPARC mono, SPARC m) and/or anti-SPARC polyclonal (SPARCpoly, SPARC p) antibodies. As described herein, the molecularcharacteristics of the tumor determined can be determined by IHCcombined with one or more of gene copy number, gene expression, andmutation analysis. The genes and/or gene products used for IHC analysiscan be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50,60, 70, 80, 90, 100 or all of the genes and/or gene products listed inTable 2.

In some embodiments, the genes used for gene expression profilingcomprise one or more of: EGFR, SPARC, C-kit, ER, PR, Androgen receptor,PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, Thymidylatesynthase, Her2/neu, TOPO2A, ADA, AR, ASNA, BCL2, BRCA2, CD33, CDW52,CES2, DNMT1, EGFR, ERBB2, ERCC3, ESR1, FOLR2, GART, GSTP1, HDAC1, HIF1A,HSPCA, IL2RA, KIT, MLH1, MS4A1, MASH2, NFKB2, NFKBIA, OGFR, PDGFC,PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA, RXRB, SPARC, SSTR1,TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGF, VHL, and ZAP70.One or more of the following genes can also be assessed by geneexpression profiling: ALK, EML4, hENT-1, IGF-1R, HSP90AA1, MMR, p16,p21, p27, PARP-1, PI3K and TLE3. The gene expression profiling can beperformed using a low density microarray, an expression microarray, acomparative genomic hybridization (CGH) microarray, a single nucleotidepolymorphism (SNP) microarray, a proteomic array an antibody array, orother array as disclosed herein or known to those of skill in the art.In some embodiments, high throughput expression arrays are used. Suchsystems include without limitation commercially available systems fromAffymetrix, Agilent or Illumina, as described in more detail herein.Expression profiling can be performed using PCR, e.g., real-time PCR(qPCR or RT-PCR). Alternate gene expression techniques can be used aswell. The genes and/or gene products examined gene expression profilinganalysis can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,40, 50, 60, 70, 80, 90, 100 or all of the genes and/or gene productslisted in Table 2.

ISH analysis can be used to profile one or more of HER2, CMET, PIK3CA,EGFR, TOP2A, CMYC and EML4-ALK fusion. ISH may include FISH, CISH or thelike. In some embodiments, ISH is used to detect or test for one or moreof the following genes, including without limitation: EGFR, SPARC,C-kit, ER, PR, AR, PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK,ERCC1, TS, HER2, or TOPO2A. In some embodiments, ISH is used to detector test for one or more of EML4-ALK fusion and IGF-1R. In someembodiments, ISH is used to detect or test various biomarkers, includingwithout limitation one or more of the following: ADA, AR, ASNA, BCL2,BRCA2, c-Met, CD33, CDW52, CES2, DNMT1, EGFR, EML4-ALK fusion, ERBB2,ERCC3, ESR1, FOLR2, GART, GSTP1, HDAC1, hENT-1, HIF1A, HSPCA, IGF-1R,IL2RA, KIT, MLH1, MMR, MS4A1, MASH2, NFKB2, NFKBIA, OGFR, p16, p21, p27,PARP-1, PI3K, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA,RXRB, SPARC, SSTR1, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS,VDR, VEGF, VHL, or ZAP70. The genes and/or gene products used for ISHanalysis can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,40, 50, 60, 70, 80, 90, 100 or all of the genes and/or gene productslisted in Table 2.

Mutation profiling can be determined by sequencing, including Sangersequencing, array sequencing, pyrosequencing, NextGen sequencing, etc.Sequence analysis may reveal that genes harbor activating mutations sothat drugs that inhibit activity are indicated for treatment.Alternately, sequence analysis may reveal that genes harbor mutationsthat inhibit or eliminate activity, thereby indicating treatment forcompensating therapies. In embodiments, sequence analysis comprises thatof exon 9 and 11 of c-KIT. Sequencing may also be performed onEGFR-kinase domain exons 18, 19, 20, and 21. Mutations, amplificationsor misregulations of EGFR or its family members are implicated in about30% of all epithelial cancers. Sequencing can also be performed on PI3K,encoded by the PIK3CA gene. This gene is a found mutated in manycancers. Sequencing analysis can also comprise assessing mutations inone or more ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2,CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR,EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1,HCK, HDAC1, HIF1A, HSP9OAA1, IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, KIT,LCK, LYN, MET, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, NFKBIA, NRAS,OGFR, PARP1, PDGFC, PDGFRA, PDGFRB, PGP, PGR, POLA1, PTEN, PTGS2,PTPN12, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC,SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1,TYMS, VDR, VEGFA, VHL, YES1, and ZAP70. One or more of the followinggenes can also be assessed by sequence analysis: ALK, EML4, hENT-1,IGF-1R, HSP90AA1, MMR, p16, p21, p27, PARP-1, PI3K and TLE3. The genesand/or gene products used for mutation or sequence analysis can be atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80,90, 100 or all of the genes and/or gene products listed in Table 2,Tables 6-9 or Tables 12-15.

In embodiments, the methods of the invention are used detect genefusions, such as those listed in U.S. patent application Ser. No.12/658,770, filed Feb. 12, 2010; International PCT Patent ApplicationPCT/US2010/000407, filed Feb. 11, 2010; and International PCT PatentApplication PCT/US2010/54366, filed Oct. 27, 2010; all of whichapplications are incorporated by reference herein in their entirety. Afusion gene is a hybrid gene created by the juxtaposition of twopreviously separate genes. This can occur by chromosomal translocationor inversion, deletion or via trans-splicing. The resulting fusion genecan cause abnormal temporal and spatial expression of genes, leading toabnormal expression of cell growth factors, angiogenesis factors, tumorpromoters or other factors contributing to the neoplastic transformationof the cell and the creation of a tumor. For example, such fusion genescan be oncogenic due to the juxtaposition of: 1) a strong promoterregion of one gene next to the coding region of a cell growth factor,tumor promoter or other gene promoting oncogenesis leading to elevatedgene expression, or 2) due to the fusion of coding regions of twodifferent genes, giving rise to a chimeric gene and thus a chimericprotein with abnormal activity. Fusion genes are characteristic of manycancers. Once a therapeutic intervention is associated with a fusion,the presence of that fusion in any type of cancer identifies thetherapeutic intervention as a candidate therapy for treating the cancer.

The presence of fusion genes, e.g., those described in U.S. patentapplication Ser. No. 12/658,770, filed Feb. 12, 2010; International PCTPatent Application PCT/US2010/000407, filed Feb. 11, 2010; andInternational PCT Patent Application PCT/US2010/54366, filed Oct. 27,2010 or elsewhere herein, can be used to guide therapeutic selection.For example, the BCR-ABL gene fusion is a characteristic molecularaberration in ˜90% of chronic myelogenous leukemia (CML) and in a subsetof acute leukemias (Kurzrock et al., Annals of Internal Medicine 2003;138:819-830). The BCR-ABL results from a translocation betweenchromosomes 9 and 22, commonly referred to as the Philadelphiachromosome or Philadelphia translocation. The translocation bringstogether the 5′ region of the BCR gene and the 3′ region of ABL1,generating a chimeric BCR-ABL1 gene, which encodes a protein withconstitutively active tyrosine kinase activity (Mittleman et al., NatureReviews Cancer 2007; 7:233-245). The aberrant tyrosine kinase activityleads to de-regulated cell signaling, cell growth and cell survival,apoptosis resistance and growth factor independence, all of whichcontribute to the pathophysiology of leukemia (Kurzrock et al., Annalsof Internal Medicine 2003; 138:819-830). Patients with the Philadelphiachromosome are treated with imatinib and other targeted therapies.Imatinib binds to the site of the constitutive tyrosine kinase activityof the fusion protein and prevents its activity. Imatinib treatment hasled to molecular responses (disappearance of BCR-ABL+ blood cells) andimproved progression-free survival in BCR-ABL+CML patients (Kantarjianet al., Clinical Cancer Research 2007; 13:1089-1097).

Another fusion gene, IGH-MYC, is a defining feature of ˜80% of Burkitt'slymphoma (Ferry et al. Oncologist 2006; 11:375-83). The causal event forthis is a translocation between chromosomes 8 and 14, bringing the c-Myconcogene adjacent to the strong promoter of the immunoglobulin heavychain gene, causing c-myc overexpression (Mittleman et al., NatureReviews Cancer 2007; 7:233-245). The c-myc rearrangement is a pivotalevent in lymphomagenesis as it results in a perpetually proliferativestate. It has wide ranging effects on progression through the cellcycle, cellular differentiation, apoptosis, and cell adhesion (Ferry etal. Oncologist 2006; 11:375-83).

A number of recurrent fusion genes have been catalogued in the Mittlemandatabase (cgap.nci.nih.gov/Chromosomes/Mitelman). The gene fusions canbe used to characterize neoplasms and cancers and guide therapy usingthe subject methods described herein. For example, TMPRSS2-ERG,TMPRSS2-ETV and SLC45A3-ELK4 fusions can be detected to characterizeprostate cancer; and ETV6-NTRK3 and ODZ4-NRG1 can be used tocharacterize breast cancer. The EML4-ALK, RLF-MYCL1, TGF-ALK, orCD74-ROS1 fusions can be used to characterize a lung cancer. TheACSL3-ETV1, C150RF21-ETV1, FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG,TMPRSS2-ETV1/4/5, TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5or KLK2-ETV4 fusions can be used to characterize a prostate cancer. TheGOPC-ROS1 fusion can be used to characterize a brain cancer. TheCHCHD7-PLAG1, CTNNB1-PLAG1, FHIT-HMGA2, HMGA2-NFIB, LIFR-PLAG1, orTCEA1-PLAG1 fusions can be used to characterize a head and neck cancer.The ALPHA-TFEB, NONO-TFE3, PRCC-TFE3, SFPQ-TFE3, CLTC-TFE3, orMALAT1-TFEB fusions can be used to characterize a renal cell carcinoma(RCC). The AKAP9-BRAF, CCDC6-RET, ERC1-RE™, GOLGA5-RET, HOOK3-RET,HRH4-RET, KTN1-RET, NCOA4-RET, PCM1-RET, PRKARA1A-RET, RFG-RET,RFG9-RET, Ria-RET, TGF-NTRK1, TPM3-NTRK1, TPM3-TPR, TPR-MET, TPR-NTRK1,TRIM24-RET, TRIM27-RET or TRIM33-RET fusions can be used to characterizea thyroid cancer and/or papillary thyroid carcinoma; and the PAX8-PPARyfusion can be analyzed to characterize a follicular thyroid cancer.Fusions that are associated with hematological malignancies includewithout limitation TTL-ETV6, CDK6-MLL, CDK6-TLX3, ETV6-FLT3, ETV6-RUNX1,ETV6-TTL, MLL-AFF1, MLL-AFF3, MLL-AFF4, MLL-GAS7, TCBA1-ETV6, TCF3-PBX1or TCF3-TFPT, which are characteristic of acute lymphocytic leukemia(ALL); BCL11B-TLX3, IL2-TNFRFS17, NUP214-ABL1, NUP98-CCDC28A, TAL1-STIL,or ETV6-ABL2, which are characteristic of T-cell acute lymphocyticleukemia (T-ALL); ATIC-ALK, KIAA1618-ALK, MSN-ALK, MYH9-ALK, NPM1-ALK,TGF-ALK or TPM3-ALK, which are characteristic of anaplastic large celllymphoma (ALCL); BCR-ABL1, BCR-JAK2, ETV6-EVI1, ETV6-MN1 or ETV6-TCBA1,characteristic of chronic myelogenous leukemia (CML); CBFB-MYH11,CHIC2-ETV6, ETV6-ABL1, ETV6-ABL2, ETV6-ARNT, ETV6-CDX2, ETV6-HLXB9,ETV6-PER1, MEF2D-DAZAP1, AML-AFF1, MLL-ARHGAP26, MLL-ARHGEF12,MLL-CASC5, MLL-CBL, MLL-CREBBP, MLL-DAB21P, MLL-ELL, MLL-EP300,MLL-EPS15, MLL-FNBP1, MLL-FOXO3A, MLL-GMPS, MLL-GPHN, MLL-MLLT1,MLL-MLLT11, MLL-MLLT3, MLL-MLLT6, MLL-MYO1F, MLL-PICALM, MLL-SEPT2,MLL-SEPT6, MLL-SORBS2, MYST3-SORBS2, MYST-CREBBP, NPM1-MLF1,NUP98-HOXA13, PRDM16-EVI1, RABEP1-PDGFRB, RUNX1-EVI1, RUNX1-MDS1,RUNX1-RPL22, RUNX1-RUNX1T1, RUNX1-SH3D19, RUNX1-USP42, RUNX1-YTHDF2,RUNX1-ZNF687, or TAF15-ZNF-384, which are characteristic of acutemyeloid leukemia (AML); CCND1-FSTL3, which is characteristic of chroniclymphocytic leukemia (CLL); BCL3-MYC, MYC-BTG1, BCL7A-MYC,BRWD3-ARHGAP20 or BTG1-MYC, which are characteristic of B-cell chroniclymphocytic leukemia (B-CLL); CITTA-BCL6, CLTC-ALK, IL21R-BCL6,PIM1-BCL6, TFCR-BCL6, IKZF1-BCL6 or SEC31A-ALK, which are characteristicof diffuse large B-cell lymphomas (DLBCL); FLIP1-PDGFRA, FLT3-ETV6,KIAA1509-PDGFRA, PDE4DIP-PDGFRB, NIN-PDGFRB, TP53BP1-PDGFRB, orTPM3-PDGFRB, which are characteristic of hyper eosinophilia/chroniceosinophilia; and IGH-MYC or LCP1-BCL6, which are characteristic ofBurkitt's lymphoma. One of skill will understand that additionalfusions, including those yet to be identified to date, can be used toguide treatment once their presence is associated with a therapeuticintervention.

The fusion genes and gene products can be detected using one or moretechniques described herein. In some embodiments, the sequence of thegene or corresponding mRNA is determined, e.g., using Sanger sequencing,NextGen sequencing, pyrosequencing, DNA microarrays, etc. Chromosomalabnormalities can be assessed using FISH or PCR techniques, amongothers. For example, a break apart probe can be used for FISH detectionof ALK fusions such as EML4-ALK, KIF5B-ALK and/or TFG-ALK. As analternate, PCR can be used to amplify the fusion product, whereinamplification or lack thereof indicates the presence or absence of thefusion, respectively. In some embodiments, the fusion protein fusion isdetected. Appropriate methods for protein analysis include withoutlimitation mass spectroscopy, electrophoresis (e.g., 2D gelelectrophoresis or SDS-PAGE) or antibody related techniques, includingimmunoassay, protein array or immunohistochemistry. The techniques canbe combined. As a non-limiting example, indication of an ALK fusion byFISH can be confirmed for ALK expression using IHC, or vice versa.

Treatment Selection

The systems and methods allow identification of one or more therapeutictargets whose projected efficacy can be linked to therapeutic efficacy,ultimately based on the molecular profiling. Illustrative schemes forusing molecular profiling to identify a treatment regime are shown inFIGS. 2, 49A-B and 50, each of which is described in further detailherein. The invention comprises use of molecular profiling results tosuggest associations with treatment responses. In an embodiment, theappropriate biomarkers for molecular profiling are selected on the basisof the subject's tumor type. These suggested biomarkers can be used tomodify a default list of biomarkers. In other embodiments, the molecularprofiling is independent of the source material. In some embodiments,rules are used to provide the suggested chemotherapy treatments based onthe molecular profiling test results. In an embodiment, the rules aregenerated from abstracts of the peer reviewed clinical oncologyliterature. Expert opinion rules can be used but are optional. In anembodiment, clinical citations are assessed for their relevance to themethods of the invention using a hierarchy derived from the evidencegrading system used by the United States Preventive Services Taskforce.The “best evidence” can be used as the basis for a rule. The simplestrules are constructed in the format of “if biomarker positive thentreatment option one, else treatment option two.” Treatment optionscomprise no treatment with a specific drug, treatment with a specificdrug or treatment with a combination of drugs. In some embodiments, morecomplex rules are constructed that involve the interaction of two ormore biomarkers. In such cases, the more complex interactions aretypically supported by clinical studies that analyze the interactionbetween the biomarkers included in the rule. Finally, a report can begenerated that describes the association of the chemotherapy responseand the biomarker and a summary statement of the best evidencesupporting the treatments selected. Ultimately, the treating physicianwill decide on the best course of treatment.

As a non-limiting example, molecular profiling might reveal that theEGFR gene is amplified or overexpressed, thus indicating selection of atreatment that can block EGFR activity, such as the monoclonal antibodyinhibitors cetuximab and panitumumab, or small molecule kinaseinhibitors effective in patients with activating mutations in EGFR suchas gefitinib, erlotinib, and lapatinib. Other anti-EGFR monoclonalantibodies in clinical development include zalutumumab, nimotuzumab, andmatuzumab. The candidate treatment selected can depend on the settingrevealed by molecular profiling. For example, kinase inhibitors areoften prescribed with EGFR is found to have activating mutations.Continuing with the illustrative embodiment, molecular profiling mayalso reveal that some or all of these treatments are likely to be lesseffective. For example, patients taking gefitinib or erlotinibeventually develop drug resistance mutations in EGFR. Accordingly, thepresence of a drug resistance mutation would contraindicate selection ofthe small molecule kinase inhibitors. One of skill will appreciate thatthis example can be expanded to guide the selection of other candidatetreatments that act against genes or gene products whose differentialexpression is revealed by molecular profiling. Similarly, candidateagents known to be effective against diseased cells carrying certainnucleic acid variants can be selected if molecular profiling revealssuch variants.

As another example, consider the drug imatinib, currently marketed byNovartis as Gleevec in the US in the form of imatinib mesylate. Imatinibis a 2-phenylaminopyrimidine derivative that functions as a specificinhibitor of a number of tyrosine kinase enzymes. It occupies thetyrosine kinase active site, leading to a decrease in kinase activity.Imatinib has been shown to block the activity of Abelson cytoplasmictyrosine kinase (ABL), c-Kit and the platelet-derived growth factorreceptor (PDGFR). Thus, imatinib can be indicated as a candidatetherapeutic for a cancer determined by molecular profiling tooverexpress ABL, c-KIT or PDGFR. Imatinib can be indicated as acandidate therapeutic for a cancer determined by molecular profiling tohave mutations in ABL, c-KIT or PDGFR that alter their activity, e.g.,constitutive kinase activity of ABLs caused by the BCR-ABL mutation. Asan inhibitor of PDGFR, imatinib mesylate appears to have utility in thetreatment of a variety of dermatological diseases.

Cancer therapies that can be identified as candidate treatments by themethods of the invention include without limitation: 13-cis-RetinoicAcid, 2-CdA, 2-Chlorodeoxyadenosine, 5-Azacitidine, 5-Fluorouracil,5-FU, 6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine, Abraxane, Accutane®,Actinomycin-D, Adriamycin®, Adrucil®, Afinitor®, Agrylin®, Ala-Cort®,Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®, Alkeran®,All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin,Amifostine, Aminoglutethimide, Anagrelide, Anandron®, Anastrozole,Arabinosylcytosine, Ara-C, Aranesp®, Aredia®, Arimidex®, Aromasin®,Arranon®, Arsenic Trioxide, Asparaginase, ATRA, Avastin®, Azacitidine,BCG, BCNU, Bendamustine, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide,BiCNU, Blenoxane®, Bleomycin, Bortezomib, Busulfan, Busulfex®, C225,Calcium Leucovorin, Campath®, Camptosar®, Camptothecin-11, Capecitabine,Carac™, Carboplatin, Carmustine, Carmustine Wafer, Casodex®, CC-5013,CCI-779, CCNU, CDDP, CeeNU, Cerubidine®, Cetuximab, Chlorambucil,Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen®, CPT-11,Cyclophosphamide, Cytadren®, Cytarabine, Cytarabine Liposomal,Cytosar-U®, Cytoxan®, Dacarbazine, Dacogen, Dactinomycin, DarbepoetinAlfa, Dasatinib, Daunomycin Daunorubicin, Daunorubicin Hydrochloride,Daunorubicin Liposomal, DaunoXome®, Decadron, Decitabine, Delta-Cortef®,Deltasone®, Denileukin, Diftitox, DepoCyt™ Dexamethasone, DexamethasoneAcetate Dexamethasone Sodium Phosphate, Dexasone, Dexrazoxane, DHAD,DIC, Diodex Docetaxel, Doxorubicin, Doxorubicin Liposomal, Droxia™,DTIC, DTIC-Dome®, Duralone®, Efudex®, Eligard™, Ellence™, Eloxatin™,Elspar®, Emcyt®, Epirubicin, Epoetin Alfa, Erbitux, Erlotinib, ErwiniaL-asparaginase, Estramustine, Ethyol Etopophos®, Etoposide, EtoposidePhosphate, Eulexin®, Everolimus, Evista®, Exemestane, Fareston®,Faslodex®, Femara®, Filgrastim, Floxuridine, Fludara®, Fludarabine,Fluoroplex®, Fluorouracil, Fluorouracil (cream), Fluoxymesterone,Flutamide, Folinic Acid, FUDR®, Fulvestrant, G-CSF, Gefitinib,Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec™, Gliadel® Wafer,GM-CSF, Goserelin, Granulocyte—Colony Stimulating Factor, GranulocyteMacrophage Colony Stimulating Factor, Halotestin®, Herceptin®, Hexadrol,Hexalen®, Hexamethylmelamine, HMM, Hycamtin®, Hydrea®, HydrocortAcetate®, Hydrocortisone, Hydrocortisone Sodium Phosphate,Hydrocortisone Sodium Succinate, Hydrocortone Phosphate, Hydroxyurea,Ibritumomab, Ibritumomab, Tiuxetan, Idamycin®, Idarubicin, Ifex®,IFN-alpha, Ifosfamide, IL-11, IL-2, Imatinib mesylate, ImidazoleCarboxamide, Interferon alfa, Interferon Alfa-2b (PEG Conjugate),Interleukin-2, Interleukin-11, Intron A® (interferon alfa-2b), Iressa®,Irinotecan, Isotretinoin, Ixabepilone, Ixempra™ Kidrolase (t),Lanacort®, Lapatinib, L-asparaginase, LCR, Lenalidomide, Letrozole,Leucovorin, Leukeran, Leukine™, Leuprolide, Leurocristine, Leustatin™,Liposomal Ara-C Liquid Pred®, Lomustine, L-PAM, L-Sarcolysin, Lupron®,Lupron Depot®, Matulane®, Maxidex, Mechlorethamine, MechlorethamineHydrochloride, Medralone®, Medrol®, Megace®, Megestrol, MegestrolAcetate, Melphalan, Mercaptopurine, Mesna, Mesnex™, Methotrexate,Methotrexate Sodium, Methylprednisolone, Meticorten®, Mitomycin,Mitomycin-C, Mitoxantrone, M-Prednisol®, MTC, MTX, Mustargen®, Mustine,Mutamycin®, Myleran®, Mylocel™, Mylotargt, Navelbine®, Nelarabine,Neosar®, Neulasta™, Neumegat, Neupogen®, Nexavart, Nilandron®,Nilutamide, Nipent®, Nitrogen Mustard, Novaldext, Novantrone®,Octreotide, Octreotide acetate, Oncospart, Oncovin®, Ontakt, Onxal™,Oprevelkin, Orapredt, Orasone®, Oxaliplatin, Paclitaxel, PaclitaxelProtein-bound, Pamidronate, Panitumumab, Panretint, Paraplatin®,Pediapred®, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON™,PEG-L-asparaginase, PEMETREXED, Pentostatin, Phenylalanine Mustard,Platinol®, Platinol-AQ®, Prednisolone, Prednisone, Prelone®,Procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with CarmustineImplant, Purinethol®, Raloxifene, Revlimidt, Rheumatrex®, Rituxant,Rituximab, Roferon-A® (Interferon Alfa-2a), Rubex®, Rubidomycinhydrochloride, Sandostatin®, Sandostatin LAR®, Sargramostim,Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, STI-571, Streptozocin,SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretint, Taxol®,Taxotere®, Temodart, Temozolomide, Temsirolimus, Teniposide, TESPA,Thalidomide, Thalomidt, TheraCys®, Thioguanine, Thioguanine Tabloid®,Thiophosphoamide, Thioplext, Thiotepa, TICE®, Toposart, Topotecan,Toremifene, Toriselt, Tositumomab, Trastuzumab, Treandat, Tretinoin,Trexall™, Trisenoxt, TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcadet,VePesid®, Vesanoidt, Viadur™, Vidazat, Vinblastine, Vinblastine Sulfate,Vincasar Pfs®, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB,VM-26, Vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, Zevalin™,Zinecardt, Zoladex®, Zoledronic acid, Zolinza, Zometat, and anyappropriate combinations thereof.

The candidate treatments identified according to the subject methods canbe chosen from the class of therapeutic agents identified asAnthracyclines and related substances, Anti-androgens, Anti-estrogens,Antigrowth hormones (e.g., Somatostatin analogs), Combination therapy(e.g., vincristine, bcnu, melphalan, cyclophosphamide, prednisone(VBMCP)), DNA methyltransferase inhibitors, Endocrine therapy—Enzymeinhibitor, Endocrine therapy—other hormone antagonists and relatedagents, Folic acid analogs (e.g., methotrexate), Folic acid analogs(e.g., pemetrexed), Gonadotropin releasing hormone analogs,Gonadotropin-releasing hormones, Monoclonal antibodies(EGFR-Targeted—e.g., panitumumab, cetuximab), Monoclonal antibodies(Her2-Targeted—e.g., trastuzumab), Monoclonal antibodies(Multi-Targeted—e.g., alemtuzumab), Other alkylating agents, Otherantineoplastic agents (e.g., asparaginase), Other antineoplastic agents(e.g., ATRA), Other antineoplastic agents (e.g., bexarotene), Otherantineoplastic agents (e.g., celecoxib), Other antineoplastic agents(e.g., gemcitabine), Other antineoplastic agents (e.g., hydroxyurea),Other antineoplastic agents (e.g., irinotecan, topotecan), Otherantineoplastic agents (e.g., pentostatin), Other cytotoxic antibiotics,Platinum compounds, Podophyllotoxin derivatives (e.g., etoposide),Progestogens, Protein kinase inhibitors (EGFR-Targeted), Protein kinaseinhibitors (Her2 targeted therapy—e.g., lapatinib), Pyrimidine analogs(e.g., cytarabine), Pyrimidine analogs (e.g., fluoropyrimidines),Salicylic acid and derivatives (e.g., aspirin), Src-family proteintyrosine kinase inhibitors (e.g., dasatinib), Taxanes, Taxanes (e.g.,nab-paclitaxel), Vinca Alkaloids and analogs, Vitamin D and analogs,Monoclonal antibodies (Multi-Targeted—e.g., bevacizumab), Protein kinaseinhibitors (e.g., imatinib, sorafenib, sunitinib), Tyrosine Kinaseinhibitors (TKI) (e.g., vemurafenib, sorafenib, imatinib, sunitinib,erlotinib, gefitinib, crizotinib, lapatinib).

In some embodiments, the candidate treatments identified according tothe subject methods are chosen from at least the groups of treatmentsconsisting of 5-fluorouracil, abarelix, alemtuzumab, aminoglutethimide,anastrozole, asparaginase, aspirin, ATRA, azacitidine, bevacizumab,bexarotene, bicalutamide, calcitriol, capecitabine, carboplatin,celecoxib, cetuximab, chemotherapy, cholecalciferol, cisplatin,cytarabine, dasatinib, daunorubicin, decitabine, doxorubicin,epirubicin, erlotinib, etoposide, exemestane, flutamide, fulvestrant,gefitinib, gemcitabine, gonadorelin, goserelin, hydroxyurea, imatinib,irinotecan, lapatinib, letrozole, leuprolide, liposomal-doxorubicin,medroxyprogesterone, megestrol, megestrol acetate, methotrexate,mitomycin, nab-paclitaxel, octreotide, oxaliplatin, paclitaxel,panitumumab, pegaspargase, pemetrexed, pentostatin, sorafenib,sunitinib, tamoxifen, Taxanes, temozolomide, toremifene, trastuzumab,VBMCP, and vincristine. The candidate treatments can be any of those inany one of Tables 3-6, Tables 9-10, Table 17, and Tables 22-24 herein.

Rules Engine

In some embodiments, a database is created that maps treatments andmolecular profiling results. The treatment information can include theprojected efficacy of a therapeutic agent against cells having certainattributes that can be measured by molecular profiling. The molecularprofiling can include differential expression or mutations in certaingenes, proteins, or other biological molecules of interest. Through themapping, the results of the molecular profiling can be compared againstthe database to select treatments. The database can include bothpositive and negative mappings between treatments and molecularprofiling results. In some embodiments, the mapping is created byreviewing the literature for links between biological agents andtherapeutic agents. For example, a journal article, patent publicationor patent application publication, scientific presentation, etc can bereviewed for potential mappings. The mapping can include results of invivo, e.g., animal studies or clinical trials, or in vitro experiments,e.g., cell culture. Any mappings that are found can be entered into thedatabase, e.g., cytotoxic effects of a therapeutic agent against cellsexpressing a gene or protein. In this manner, the database can becontinuously updated. It will be appreciated that the methods of theinvention are updated as well.

The rules can be generated by evidence-based literature review.Biomarker research continues to provide a better understanding of theclinical behavior and biology of cancer. This body of literature can bemaintained in an up-to-date data repository incorporating recentclinical studies relevant to treatment options and potential clinicaloutcomes. The studies can be ranked so that only those with thestrongest or most reliable evidence are selected for rules generation.For example, the rules generation can employ the grading system from thecurrent methods of the U.S. Preventive Services Task Force. Theliterature evidence can be reviewed and evaluated based on the strengthof clinical evidence supporting associations between biomarkers andtreatments in the literature study. This process can be performed by astaff of scientists, physicians and other skilled reviewers. The processcan also be automated in whole or in part by using language search andheuristics to identify relevant literature. The rules can be generatedby a review of a plurality of literature references, e.g., tens,hundreds, thousands or more literature articles.

In another aspect, the invention provides a method of generating a setof evidence-based associations, comprising: (a) searching one or moreliterature database by a computer using an evidence-based medicinesearch filter to identify articles comprising a gene or gene productthereof, a disease, and one or more therapeutic agent; (b) filtering thearticles identified in (a) to compile evidence-based associationscomprising the expected benefit and/or the expected lack of benefit ofthe one or more therapeutic agent for treating the disease given thestatus of the gene or gene product; (c) adding the evidence-basedassociations compiled in (b) to the set of evidence-based associations;and (d) repeating steps (a)-(c) for an additional gene or gene productthereof. The status of the gene can include one or more assessments asdescribed herein which relate to a biological state, e.g., one or moreof an expression level, a copy number, and a mutation. The genes or geneproducts thereof can be one or more genes or gene products thereofselected from Table 2, Tables 6-9 or Tables 12-15. For example, themethod can be repeated for at least 1, e.g., at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600 or at least 700 of the genes or gene products thereof in Table2, Tables 6-9 or Tables 12-15. The disease can be a disease describedhere, e.g., in embodiment the disease comprises a cancer. The one ormore literature database can be selected from the group consisting ofthe National Library of Medicine's (NLM's) MEDLINE™ database ofcitations, a patent literature database, and a combination thereof.

Evidence-based medicine (EBM) or evidence-based practice (EBP) aims toapply the best available evidence gained from the scientific method toclinical decision making. This approach assesses the strength ofevidence of the risks and benefits of treatments (including lack oftreatment) and diagnostic tests. Evidence quality can be assessed basedon the source type (from meta-analyses and systematic reviews ofdouble-blind, placebo-controlled clinical trials at the top end, down toconventional wisdom at the bottom), as well as other factors includingstatistical validity, clinical relevance, currency, and peer-reviewacceptance. Evidence-based medicine filters are searches that have beendeveloped to facilitate searches in specific areas of clinical medicinerelated to evidence-based medicine (diagnosis, etiology, meta-analysis,prognosis and therapy). They are designed to retrieve high qualityevidence from published studies appropriate to decision-making. Theevidence-based medicine filter used in the invention can be selectedfrom the group consisting of a generic evidence-based medicine filter, aMcMaster University optimal search strategy evidence-based medicinefilter, a University of York statistically developed searchevidence-based medicine filter, and a University of California SanFrancisco systemic review evidence-based medicine filter. See e.g., USPatent Publication 20080215570; Shojania and Bero. Taking advantage ofthe explosion of systematic reviews: an efficient MEDLINE searchstrategy. Eff Clin Pract. 2001 July-August; 4(4):157-62; Ingui andRogers. Searching for clinical prediction rules in MEDLINE. J Am MedInform Assoc. 2001 July-August; 8(4):391-7; Haynes et al., Optimalsearch strategies for retrieving scientifically strong studies oftreatment from Medline: analytical survey. BMJ. 2005 May 21;330(7501):1179; Wilczynski and Haynes. Consistency and accuracy ofindexing systematic review articles and meta-analyses in medline. HealthInfo Libr J. 2009 September; 26(3):203-10; which references areincorporated by reference herein in their entirety. A generic filter canbe a customized filter based on an algorithm to identify the desiredreferences from the one or more literature database. For example, themethod can use one or more approach as described in U.S. Pat. No.5,168,533 to Kato et al., U.S. Pat. No. 6,886,010 to Kostoff, or USPatent Application Publication No. 20040064438 to Kostoff; whichreferences are incorporated by reference herein in their entirety.

The further filtering of articles identified by the evidence-basedmedicine filter can be performed using a computer, by one or more expertuser, or combination thereof. The one or more expert can be a trainedscientist or physician. In embodiments, the set of evidence-basedassociations comprise one or more of the rules in any of Tables 3-6,Tables 9-10, Table 17, and Tables 22-24. For example, the set ofevidence-based associations can include at least 5, 10, 25, 50 or 100rules in Tables 3-6, Tables 9-10, Table 17, and Tables 22-24. In someembodiments, the set of evidence-based associations comprises orconsists of all of the rules in any of Tables 3-6, Tables 9-10, Table17, and Tables 22-24. In an aspect, the invention provides a computerreadable medium comprising the set of evidence-based associationsgenerated by the subject methods. The invention further provides acomputer readable medium comprising one or more rules in any of Tables3-6, Tables 9-10, Table 17, and Tables 22-24 herein. In an embodiment,the computer readable medium comprises at least 5, 10, 25, 50 or 100rules in any of Tables 3-6, Tables 9-10, Table 17, and Tables 22-24. Forexample, the computer readable medium can comprise all rules in any ofTables 3-6, Tables 9-10, Table 17, and Tables 22-24, e.g., all rules inTables 3-6, Tables 9-10, Table 17, and Tables 22-24.

The rules for the mappings can contain a variety of supplementalinformation. In some embodiments, the database contains prioritizationcriteria. For example, a treatment with more projected efficacy in agiven setting can be preferred over a treatment projected to have lesserefficacy. A mapping derived from a certain setting, e.g., a clinicaltrial, may be prioritized over a mapping derived from another setting,e.g., cell culture experiments. A treatment with strong literaturesupport may be prioritized over a treatment supported by morepreliminary results. A treatment generally applied to the type ofdisease in question, e.g., cancer of a certain tissue origin, may beprioritized over a treatment that is not indicated for that particulardisease. Mappings can include both positive and negative correlationsbetween a treatment and a molecular profiling result. In a non-limitingexample, one mapping might suggest use of a kinase inhibitor likeerlotinib against a tumor having an activating mutation in EGFR, whereasanother mapping might suggest against that treatment if the EGFR alsohas a drug resistance mutation. Similarly, a treatment might beindicated as effective in cells that overexpress a certain gene orprotein but indicated as not effective if the gene or protein isunderexpressed.

The selection of a candidate treatment for an individual can be based onmolecular profiling results from any one or more of the methodsdescribed. Alternatively, selection of a candidate treatment for anindividual can be based on molecular profiling results from more thanone of the methods described. For example, selection of treatment for anindividual can be based on molecular profiling results from FISH alone,IHC alone, or microarray analysis alone. In other embodiments, selectionof treatment for an individual can be based on molecular profilingresults from IHC, FISH, and microarray analysis; IHC and FISH; IHC andmicroarray analysis, or FISH and microarray analysis. Selection oftreatment for an individual can also be based on molecular profilingresults from sequencing or other methods of mutation detection.Molecular profiling results may include mutation analysis along with oneor more methods, such as IHC, immunoassay, and/or microarray analysis.Different combinations and sequential results can be used. For example,treatment can be prioritized according the results obtained by molecularprofiling. In an embodiment, the prioritization is based on thefollowing algorithm: 1) IHC/FISH and microarray indicates same target asa first priority; 2) IHC positive result alone next priority; or 3)microarray positive result alone as last priority. Sequencing can alsobe used to guide selection. In some embodiments, sequencing reveals adrug resistance mutation so that the effected drug is not selected evenif techniques including IHC, microarray and/or FISH indicatedifferential expression of the target molecule. Any suchcontraindication, e.g., differential expression or mutation of anothergene or gene product may override selection of a treatment.

An illustrative listing of microarray expression results versuspredicted treatments is presented in Table 3. As disclosed herein,molecular profiling is performed to determine whether a gene or geneproduct is differentially expressed in a sample as compared to acontrol. The expression status of the gene or gene product is used toselect agents that are predicted to be efficacious or not. For example,Table 3 shows that overexpression of the ADA gene or protein points topentostatin as a possible treatment. On the other hand, underexpressionof the ADA gene or protein implicates resistance to cytarabine,suggesting that cytarabine is not an optimal treatment.

TABLE 3 Molecular Profiling Results and Predicted Treatments Gene NameExpression Status Candidate Agent(s) Possible Resistance ADAOverexpressed pentostatin ADA Underexpressed cytarabine AR Overexpressedabarelix, bicalutamide, flutamide, gonadorelin, goserelin, leuprolideASNS Underexpressed asparaginase, pegaspargase BCRP (ABCG2)Overexpressed cisplatin, carboplatin, irinotecan, topotecan BRCA1Underexpressed mitomycin BRCA2 Underexpressed mitomycin CD52Overexpressed alemtuzumab CDA Overexpressed cytarabine CES2Overexpressed irinotecan c-kit Overexpressed sorafenib, sunitinib,imatinib COX-2 Overexpressed celecoxib DCK Overexpressed gemcitabinecytarabine DHFR Underexpressed methotrexate, pemetrexed DHFROverexpressed methotrexate DNMT1 Overexpressed azacitidine, decitabineDNMT3A Overexpressed azacitidine, decitabine DNMT3B Overexpressedazacitidine, decitabine EGFR Overexpressed erlotinib, gefitinib,cetuximab, panitumumab EML4-ALK Overexpressed (present) crizotinib EPHA2Overexpressed dasatinib ER Overexpressed anastrazole, exemestane,fulvestrant, letrozole, megestrol, tamoxifen, medroxyprogesterone,toremifene, aminoglutethimide ERCC1 Overexpressed carboplatin, cisplatinGART Underexpressed pemetrexed HER-2 (ERBB2) Overexpressed trastuzumab,lapatinib HIF-1α Overexpressed sorafenib, sunitinib, bevacizumab IκB-αOverexpressed bortezomib MGMT Underexpressed temozolomide MGMTOverexpressed temozolomide MRP1 (ABCC1) Overexpressed etoposide,paclitaxel, docetaxel, vinblastine, vinorelbine, topotecan, teniposideP-gp (ABCB1) Overexpressed doxorubicin, etoposide, epirubicin,paclitaxel, docetaxel, vinblastine, vinorelbine, topotecan, teniposide,liposomal doxorubicin PDGFR-α Overexpressed sorafenib, sunitinib,imatinib PDGFR-β Overexpressed sorafenib, sunitinib, imatinib PROverexpressed exemestane, fulvestrant, gonadorelin, goserelin,medroxyprogesterone, megestrol, tamoxifen, toremifene RARA OverexpressedATRA RRM1 Underexpressed gemcitabine, hydroxyurea RRM2 Underexpressedgemcitabine, hydroxyurea RRM2B Underexpressed gemcitabine, hydroxyureaRXR-α Overexpressed bexarotene RXR-β Overexpressed bexarotene SPARCOverexpressed nab-paclitaxel SRC Overexpressed dasatinib SSTR2Overexpressed octreotide SSTR5 Overexpressed octreotide TOPO IOverexpressed irinotecan, topotecan TOPO IIα Overexpressed doxorubicin,epirubicin, liposomal-doxorubicin TOPO IIβ Overexpressed doxorubicin,epirubicin, liposomal- doxorubicin TS Underexpressed capecitabine, 5-fluorouracil, pemetrexed TS Overexpressed capecitabine, 5- fluorouracilVDR Overexpressed calcitriol, cholecalciferol VEGFR1 (Flt1)Overexpressed sorafenib, sunitinib, bevacizumab VEGFR2 Overexpressedsorafenib, sunitinib, bevacizumab VHL Underexpressed sorafenib,sunitinib

Table 4 presents a selection of illustrative rules for treatmentselection. The table is ordered by groups of related therapeutic agents.Each row describes a rule that maps the information derived frommolecular profiling with an indication of benefit or lack of benefit forthe therapeutic agent. Thus, the database contains a mapping oftreatments whose biological activity is known against cancer cells thathave alterations in certain genes or gene products, including gene copyalterations, chromosomal abnormalities, overexpression of orunderexpression of one or more genes or gene products, or have variousmutations. For each agent, a Lineage is presented as applicable whichcorresponds to a type of cancer associated with use of the agent. Inthis example, the agents can be used for all cancers. Agents withBenefit are listed along with a Benefit Summary Statement that describesmolecular profiling information that relates to the predicted beneficialagent. Similarly, agents with Lack of Benefit are listed along with aLack of Benefit Summary Statement that describes molecular profilinginformation that relates to the lack of benefit associated with theagent. Finally, the molecular profiling Criteria are shown. In thecriteria, results from analysis using DNA microarray (DMA), IHC, FISH,and mutation analysis (MA) for one or more biomarkers is listed. Formicroarray analysis, expression can be reported as over (overexpressed)or under (underexpressed). When these criteria are met according to theapplication of the molecular profiling techniques to a sample, then thetherapeutic agent or agents are predicted to have a benefit or lack ofbenefit as indicated in the corresponding row.

Further drug associations and rules that can be used in embodiments ofthe invention are found in U.S. Patent Application Publication20100304989, filed Feb. 12, 2010; International PCT Patent ApplicationWO/2010/093465, filed Feb. 11, 2010; and International PCT PatentApplication WO/2011/056688, filed Oct. 27, 2010; all of whichapplications are incorporated by reference herein in their entirety. Seee.g., “Table 4: Rules Summary for Treatment Selection” ofWO/2011/056688.

TABLE 4 Exemplary Rules Summary for Treatment Selection Agents Lack ofAgents Benefit with Benefit Therapeutic with Summary Lack of SummaryAgent Lineage Benefit Statement Benefit Statement Criteria Proteinkinase None sunitinib, Presence of c- DMA: VEGFR1 inhibitors sorafenibKit mutation in overexpressed. (imatinib, exon 9 has been DMA: HIF1Asorafenib, associated with overexpressed. sunitinib) benefit from DMA:VEGFR2 sunitinib. In overexpressed. addition, over DMA: KIT expressionof overexpressed. HIF1A, DMA: PDGFRA VEGFR1, overexpressed. VEGFR2,c-Kit, DMA: PDGFRB PDGFRA and overexpressed. PDGFRB, and DMA: VHL underunderexpressed. expression of MA: c-kit VHL have been mutated -associated with Exon 9 benefit from sunitinib and sorafenib. Proteinkinase None sunitinib, Presence of c- DMA: VEGFR1 inhibitors sorafenibKit mutation in overexpressed. (imatinib, exon 9 has been DMA: HIF1Asorafenib, associated with overexpressed. sunitinib) benefit from DMA:VEGFR2 sunitinib. In overexpressed. addition, over DMA: KIT expressionof overexpressed. HIF1A, DMA: PDGFRA VEGFR1, overexpressed. VEGFR2,c-Kit, DMA: PDGFRB PDGFRA and overexpressed. PDGFRB have DMA: VHL. MA:been associated c-kit mutated - with benefit Exon 9 from sunitinib andsorafenib. Protein kinase None sunitinib, Presence of c- DMA: VEGFR1inhibitors sorafenib Kit mutation in overexpressed. (imatinib, exon 9has been DMA: HIF1A sorafenib, associated with overexpressed. sunitinib)benefit from DMA: VEGFR2. sunitinib. In DMA: KIT addition, overoverexpressed. expression of DMA: PDGFRA HIF1A, overexpressed. VEGFR1,c-Kit, DMA: PDGFRB PDGFRA and overexpressed. PDGFRB, and DMA: VHL underunderexpressed. expression of MA: c-kit VHL have been mutated -associated with Exon 9 benefit from sunitinib and sorafenib. Proteinkinase None sunitinib, Presence of c- DMA: VEGFR1 inhibitors sorafenibKit mutation in overexpressed. (imatinib, exon 9 has been DMA: HIF1Asorafenib, associated with overexpressed. sunitinib) benefit from DMA:VEGFR2. sunitinib. In DMA: KIT addition, over overexpressed. expressionof DMA: PDGFRA HIF1A, overexpressed. VEGFR1, c-Kit, DMA: PDGFRB PDGFRAand overexpressed. PDGFRB have DMA: VHL. been associated MA: c-kit withbenefit mutated - from sunitinib Exon 9, and sorafenib. Protein kinaseNone sunitinib, Presence of c- DMA: VEGFR1. inhibitors sorafenib Kitmutation in DMA: HIF1A (imatinib, exon 9 has been overexpressed.sorafenib, associated with DMA: VEGFR2 sunitinib) benefit fromoverexpressed. sunitinib. In DMA: KIT addition, over overexpressed.expression of DMA: PDGFRA HIF1A, overexpressed. VEGFR2, c-Kit, DMA:PDGFRB PDGFRA and overexpressed. PDGFRB, and DMA: VHL underunderexpressed. expression of MA: c-kit VHL have been mutated -associated with Exon 9 benefit from sunitinib and sorafenib. Proteinkinase None sunitinib, Presence of c- DMA: VEGFR1. inhibitors sorafenibKit mutation in DMA: HIF1A (imatinib, exon 9 has been overexpressed.sorafenib, associated with DMA: VEGFR2 sunitinib) benefit fromoverexpressed. sunitinib. In DMA: KIT addition, over overexpressed.expression of DMA: PDGFRA HIF1A, overexpressed. VEGFR2, c-Kit, DMA:PDGFRB PDGFRA and overexpressed. PDGFRB have DMA: VHL. been associatedMA: c-kit with benefit mutated - from sunitinib Exon 9 and sorafenib.Protein kinase None sunitinib, Presence of c- DMA: VEGFR1. inhibitorssorafenib Kit mutation in DMA: HIF1A (imatinib, exon 9 has beenoverexpressed. sorafenib, associated with DMA: VEGFR2. sunitinib)benefit from DMA: KIT sunitinib. In overexpressed. addition, over DMA:PDGFRA expression of overexpressed. HIF1A, c-Kit, DMA: PDGFRB PDGFRA andoverexpressed. PDGFRB, and DMA: VHL under underexpressed. expression ofMA: c-kit VHL have been mutated - associated with Exon 9 benefit fromsunitinib and sorafenib. Protein kinase None sunitinib, Presence of c-DMA: VEGFR1. inhibitors sorafenib Kit mutation in DMA: HIF1A (imatinib,exon 9 has been overexpressed. sorafenib, associated with DMA: VEGFR2.sunitinib) benefit from DMA: KIT sunitinib. In overexpressed. addition,over DMA: PDGFRA expression of overexpressed. HIF1A, c-Kit, DMA: PDGFRBPDGFRA and overexpressed. PDGFRB have DMA: VHL. been associated MA:c-kit with benefit mutated - from sunitinib Exon 9 and sorafenib.Protein kinase None sunitinib, Presence of c- DMA: VEGFR1 inhibitorssorafenib Kit mutation in overexpressed. (imatinib, exon 9 has been DMA:HIF1A sorafenib, associated with overexpressed. sunitinib) benefit fromDMA: VEGFR2 sunitinib. In overexpressed. addition, over DMA: KITexpression of overexpressed. HIF1A, DMA: PDGFRA VEGFR1, overexpressed.VEGFR2, c-Kit DMA: PDGFRB. and PDGFRA, DMA: VHL and underunderexpressed. expression of MA: c-kit VHL have been mutated -associated with Exon 9 benefit from sunitinib and sorafenib.

The efficacy of various therapeutic agents given particular assayresults, such as those in Table 4 above, is derived from reviewing,analyzing and rendering conclusions on empirical evidence, such as thatis available the medical literature or other medical knowledge base. Theresults are used to guide the selection of certain therapeutic agents ina prioritized list for use in treatment of an individual. When molecularprofiling results are obtained, e.g., differential expression ormutation of a gene or gene product, the results can be compared againstthe database to guide treatment selection. The set of rules in thedatabase can be updated as new treatments and new treatment data becomeavailable. In some embodiments, the rules database is updatedcontinuously. In some embodiments, the rules database is updated on aperiodic basis. Any relevant correlative or comparative approach can beused to compare the molecular profiling results to the rules database.In one embodiment, a gene or gene product is identified asdifferentially expressed by molecular profiling. The rules database isqueried to select entries for that gene or gene product. Treatmentselection information selected from the rules database is extracted andused to select a treatment. The information, e.g., to recommend or notrecommend a particular treatment, can be dependent on whether the geneor gene product is over or underexpressed, or has other abnormalities atthe genetic or protein levels as compared to a reference. In some cases,multiple rules and treatments may be pulled from a database comprisingthe comprehensive rules set depending on the results of the molecularprofiling. In some embodiments, the treatment options are presented in aprioritized list. In some embodiments, the treatment options arepresented without prioritization information. In either case, anindividual, e.g., the treating physician or similar caregiver may choosefrom the available options.

The methods described herein are used to prolong survival of a subjectby providing personalized treatment. In some embodiments, the subjecthas been previously treated with one or more therapeutic agents to treatthe disease, e.g., a cancer. The cancer may be refractory to one ofthese agents, e.g., by acquiring drug resistance mutations. In someembodiments, the cancer is metastatic. In some embodiments, the subjecthas not previously been treated with one or more therapeutic agentsidentified by the method. Using molecular profiling, candidatetreatments can be selected regardless of the stage, anatomical location,or anatomical origin of the cancer cells.

Progression-free survival (PFS) denotes the chances of staying free ofdisease progression for an individual or a group of individualssuffering from a disease, e.g., a cancer, after initiating a course oftreatment. It can refer to the percentage of individuals in a groupwhose disease is likely to remain stable (e.g., not show signs ofprogression) after a specified duration of time. Progression-freesurvival rates are an indication of the effectiveness of a particulartreatment. Similarly, disease-free survival (DFS) denotes the chances ofstaying free of disease after initiating a particular treatment for anindividual or a group of individuals suffering from a cancer. It canrefer to the percentage of individuals in a group who are likely to befree of disease after a specified duration of time. Disease-freesurvival rates are an indication of the effectiveness of a particulartreatment. Treatment strategies can be compared on the basis of the PFSor DFS that is achieved in similar groups of patients. Disease-freesurvival is often used with the term overall survival when cancersurvival is described.

The candidate treatment selected by molecular profiling according to theinvention can be compared to a non-molecular profiling selectedtreatment by comparing the progression free survival (PFS) using therapyselected by molecular profiling (period B) with PFS for the most recenttherapy on which the patient has just progressed (period A). See FIG.28. In one setting, a PFS(B)/PFS(A) ratio ≥1.3 was used to indicate thatthe molecular profiling selected therapy provides benefit for patient(Robert Temple, Clinical measurement in drug evaluation. Edited by WuNingano and G. T. Thicker John Wiley and Sons Ltd. 1995; Von Hoff D. D.Clin Can Res. 4: 1079, 1999: Dhani et al. Clin Cancer Res. 15: 118-123,2009). Other methods of comparing the treatment selected by molecularprofiling to a non-molecular profiling selected treatment includedetermining response rate (RECIST) and percent of patients withoutprogression or death at 4 months. The term “about” as used in thecontext of a numerical value for PFS means a variation of +/−ten percent(10%) relative to the numerical value. The PFS from a treatment selectedby molecular profiling can be extended by at least 10%, 15%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or at least 90% as compared to a non-molecularprofiling selected treatment. In some embodiments, the PFS from atreatment selected by molecular profiling can be extended by at least100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or at leastabout 1000% as compared to a non-molecular profiling selected treatment.In yet other embodiments, the PFS ratio (PFS on molecular profilingselected therapy or new treatment/PFS on prior therapy or treatment) isat least about 1.3. In yet other embodiments, the PFS ratio is at leastabout 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0. In yet otherembodiments, the PFS ratio is at least about 3, 4, 5, 6, 7, 8, 9 or 10.

Similarly, the DFS can be compared in patients whose treatment isselected with or without molecular profiling. In embodiments, DFS from atreatment selected by molecular profiling is extended by at least 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% as compared to anon-molecular profiling selected treatment. In some embodiments, the DFSfrom a treatment selected by molecular profiling can be extended by atleast 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or atleast about 1000% as compared to a non-molecular profiling selectedtreatment. In yet other embodiments, the DFS ratio (DFS on molecularprofiling selected therapy or new treatment/DFS on prior therapy ortreatment) is at least about 1.3. In yet other embodiments, the DFSratio is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or2.0. In yet other embodiments, the DFS ratio is at least about 3, 4, 5,6, 7, 8, 9 or 10.

In some embodiments, the candidate treatment of the invention will notincrease the PFS ratio or the DFS ratio in the patient, neverthelessmolecular profiling provides invaluable patient benefit. For example, insome instances no preferable treatment has been identified for thepatient. In such cases, molecular profiling provides a method toidentify a candidate treatment where none is currently identified. Themolecular profiling may extend PFS, DFS or lifespan by at least 1 week,2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks,2 months, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12months, 13 months, 14 months, 15 months, 16 months, 17 months, 18months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 monthsor 2 years. The molecular profiling may extend PFS, DFS or lifespan byat least 2½ years, 3 years, 4 years, 5 years, or more. In someembodiments, the methods of the invention improve outcome so thatpatient is in remission.

The effectiveness of a treatment can be monitored by other measures. Acomplete response (CR) comprises a complete disappearance of thedisease: no disease is evident on examination, scans or other tests. Apartial response (PR) refers to some disease remaining in the body, butthere has been a decrease in size or number of the lesions by 30% ormore. Stable disease (SD) refers to a disease that has remainedrelatively unchanged in size and number of lesions. Generally, less thana 50% decrease or a slight increase in size would be described as stabledisease. Progressive disease (PD) means that the disease has increasedin size or number on treatment. In some embodiments, molecular profilingaccording to the invention results in a complete response or partialresponse. In some embodiments, the methods of the invention result instable disease. In some embodiments, the invention is able to achievestable disease where non-molecular profiling results in progressivedisease.

Computer Systems

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, forexample Setubal and Meidanis et al., Introduction to ComputationalBiology Methods (PWS Publishing Company, Boston, 1997); Salzberg,Searles, Kasif, (Ed.), Computational Methods in Molecular Biology,(Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2.sup.nd ed., 2001). See U.S.Pat. No. 6,420,108.

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention relates to embodiments that includemethods for providing genetic information over networks such as theInternet as shown in U.S. Ser. Nos. 10/197,621, 10/063,559 (U.S.Publication Number 20020183936), Ser. Nos. 10/065,856, 10/065,868,10/328,818, 10/328,872, 10/423,403, and 60/482,389. For example, one ormore molecular profiling techniques can be performed in one location,e.g., a city, state, country or continent, and the results can betransmitted to a different city, state, country or continent. Treatmentselection can then be made in whole or in part in the second location.The methods of the invention comprise transmittal of information betweendifferent locations.

Conventional data networking, application development and otherfunctional aspects of the systems (and components of the individualoperating components of the systems) may not be described in detailherein but are part of the invention. Furthermore, the connecting linesshown in the various figures contained herein are intended to representillustrative functional relationships and/or physical couplings betweenthe various elements. It should be noted that many alternative oradditional functional relationships or physical connections may bepresent in a practical system.

The various system components discussed herein may include one or moreof the following: a host server or other computing systems including aprocessor for processing digital data; a memory coupled to the processorfor storing digital data; an input digitizer coupled to the processorfor inputting digital data; an application program stored in the memoryand accessible by the processor for directing processing of digital databy the processor; a display device coupled to the processor and memoryfor displaying information derived from digital data processed by theprocessor; and a plurality of databases. Various databases used hereinmay include: patient data such as family history, demography andenvironmental data, biological sample data, prior treatment and protocoldata, patient clinical data, molecular profiling data of biologicalsamples, data on therapeutic drug agents and/or investigative drugs, agene library, a disease library, a drug library, patient tracking data,file management data, financial management data, billing data and/orlike data useful in the operation of the system. As those skilled in theart will appreciate, user computer may include an operating system(e.g., Windows NT, 95/98/2000, OS2, UNIX, Linux, Solaris, MacOS, etc.)as well as various conventional support software and drivers typicallyassociated with computers. The computer may include any suitablepersonal computer, network computer, workstation, minicomputer,mainframe or the like. User computer can be in a home ormedical/business environment with access to a network. In anillustrative embodiment, access is through a network or the Internetthrough a commercially-available web-browser software package.

As used herein, the term “network” shall include any electroniccommunications means which incorporates both hardware and softwarecomponents of such. Communication among the parties may be accomplishedthrough any suitable communication channels, such as, for example, atelephone network, an extranet, an intranet, Internet, point ofinteraction device, personal digital assistant (e.g., Palm Pilot®,Blackberry®), cellular phone, kiosk, etc.), online communications,satellite communications, off-line communications, wirelesscommunications, transponder communications, local area network (LAN),wide area network (WAN), networked or linked devices, keyboard, mouseand/or any suitable communication or data input modality. Moreover,although the system is frequently described herein as being implementedwith TCP/IP communications protocols, the system may also be implementedusing IPX, Appletalk, IP-6, NetBIOS, OSI or any number of existing orfuture protocols. If the network is in the nature of a public network,such as the Internet, it may be advantageous to presume the network tobe insecure and open to eavesdroppers. Specific information related tothe protocols, standards, and application software used in connectionwith the Internet is generally known to those skilled in the art and, assuch, need not be detailed herein. See, for example, DILIP NAIK,INTERNET STANDARDS AND PROTOCOLS (1998); JAVA 2 COMPLETE, variousauthors, (Sybex 1999); DEBORAH RAY AND ERIC RAY, MASTERING HTML 4.0(1997); and LOSHIN, TCP/IP CLEARLY EXPLAINED (1997) and DAVID GOURLEYAND BRIAN TOTTY, HTTP, THE DEFINITIVE GUIDE (2002), the contents ofwhich are hereby incorporated by reference.

The various system components may be independently, separately orcollectively suitably coupled to the network via data links whichincludes, for example, a connection to an Internet Service Provider(ISP) over the local loop as is typically used in connection withstandard modem communication, cable modem, Dish networks, ISDN, DigitalSubscriber Line (DSL), or various wireless communication methods, see,e.g., GILBERT HELD, UNDERSTANDING DATA COMMUNICATIONS (1996), which ishereby incorporated by reference. It is noted that the network may beimplemented as other types of networks, such as an interactivetelevision (ITV) network. Moreover, the system contemplates the use,sale or distribution of any goods, services or information over anynetwork having similar functionality described herein.

As used herein, “transmit” may include sending electronic data from onesystem component to another over a network connection. Additionally, asused herein, “data” may include encompassing information such ascommands, queries, files, data for storage, and the like in digital orany other form.

The system contemplates uses in association with web services, utilitycomputing, pervasive and individualized computing, security and identitysolutions, autonomic computing, commodity computing, mobility andwireless solutions, open source, biometrics, grid computing and/or meshcomputing.

Any databases discussed herein may include relational, hierarchical,graphical, or object-oriented structure and/or any other databaseconfigurations. Common database products that may be used to implementthe databases include DB2 by IBM (White Plains, N.Y.), various databaseproducts available from Oracle Corporation (Redwood Shores, Calif.),Microsoft Access or Microsoft SQL Server by Microsoft Corporation(Redmond, Wash.), or any other suitable database product. Moreover, thedatabases may be organized in any suitable manner, for example, as datatables or lookup tables. Each record may be a single file, a series offiles, a linked series of data fields or any other data structure.Association of certain data may be accomplished through any desired dataassociation technique such as those known or practiced in the art. Forexample, the association may be accomplished either manually orautomatically. Automatic association techniques may include, forexample, a database search, a database merge, GREP, AGREP, SQL, using akey field in the tables to speed searches, sequential searches throughall the tables and files, sorting records in the file according to aknown order to simplify lookup, and/or the like. The association stepmay be accomplished by a database merge function, for example, using a“key field” in pre-selected databases or data sectors.

More particularly, a “key field” partitions the database according tothe high-level class of objects defined by the key field. For example,certain types of data may be designated as a key field in a plurality ofrelated data tables and the data tables may then be linked on the basisof the type of data in the key field. The data corresponding to the keyfield in each of the linked data tables is preferably the same or of thesame type. However, data tables having similar, though not identical,data in the key fields may also be linked by using AGREP, for example.In accordance with one embodiment, any suitable data storage techniquemay be used to store data without a standard format. Data sets may bestored using any suitable technique, including, for example, storingindividual files using an ISO/IEC 7816-4 file structure; implementing adomain whereby a dedicated file is selected that exposes one or moreelementary files containing one or more data sets; using data setsstored in individual files using a hierarchical filing system; data setsstored as records in a single file (including compression, SQLaccessible, hashed vione or more keys, numeric, alphabetical by firsttuple, etc.); Binary Large Object (BLOB); stored as ungrouped dataelements encoded using ISO/IEC 7816-6 data elements; stored as ungroupeddata elements encoded using ISO/IEC Abstract Syntax Notation (ASN.1) asin ISO/IEC 8824 and 8825; and/or other proprietary techniques that mayinclude fractal compression methods, image compression methods, etc.

In one illustrative embodiment, the ability to store a wide variety ofinformation in different formats is facilitated by storing theinformation as a BLOB. Thus, any binary information can be stored in astorage space associated with a data set. The BLOB method may store datasets as ungrouped data elements formatted as a block of binary via afixed memory offset using either fixed storage allocation, circularqueue techniques, or best practices with respect to memory management(e.g., paged memory, least recently used, etc.). By using BLOB methods,the ability to store various data sets that have different formatsfacilitates the storage of data by multiple and unrelated owners of thedata sets. For example, a first data set which may be stored may beprovided by a first party, a second data set which may be stored may beprovided by an unrelated second party, and yet a third data set whichmay be stored, may be provided by a third party unrelated to the firstand second party. Each of these three illustrative data sets may containdifferent information that is stored using different data storageformats and/or techniques. Further, each data set may contain subsets ofdata that also may be distinct from other subsets.

As stated above, in various embodiments, the data can be stored withoutregard to a common format. However, in one illustrative embodiment, thedata set (e.g., BLOB) may be annotated in a standard manner whenprovided for manipulating the data. The annotation may comprise a shortheader, trailer, or other appropriate indicator related to each data setthat is configured to convey information useful in managing the variousdata sets. For example, the annotation may be called a “conditionheader”, “header”, “trailer”, or “status”, herein, and may comprise anindication of the status of the data set or may include an identifiercorrelated to a specific issuer or owner of the data. Subsequent bytesof data may be used to indicate for example, the identity of the issueror owner of the data, user, transaction/membership account identifier orthe like. Each of these condition annotations are further discussedherein.

The data set annotation may also be used for other types of statusinformation as well as various other purposes. For example, the data setannotation may include security information establishing access levels.The access levels may, for example, be configured to permit only certainindividuals, levels of employees, companies, or other entities to accessdata sets, or to permit access to specific data sets based on thetransaction, issuer or owner of data, user or the like. Furthermore, thesecurity information may restrict/permit only certain actions such asaccessing, modifying, and/or deleting data sets. In one example, thedata set annotation indicates that only the data set owner or the userare permitted to delete a data set, various identified users may bepermitted to access the data set for reading, and others are altogetherexcluded from accessing the data set. However, other access restrictionparameters may also be used allowing various entities to access a dataset with various permission levels as appropriate. The data, includingthe header or trailer may be received by a standalone interaction deviceconfigured to add, delete, modify, or augment the data in accordancewith the header or trailer.

One skilled in the art will also appreciate that, for security reasons,any databases, systems, devices, servers or other components of thesystem may consist of any combination thereof at a single location or atmultiple locations, wherein each database or system includes any ofvarious suitable security features, such as firewalls, access codes,encryption, decryption, compression, decompression, and/or the like.

The computing unit of the web client may be further equipped with anInternet browser connected to the Internet or an intranet using standarddial-up, cable, DSL or any other Internet protocol known in the art.Transactions originating at a web client may pass through a firewall inorder to prevent unauthorized access from users of other networks.Further, additional firewalls may be deployed between the varyingcomponents of CMS to further enhance security.

Firewall may include any hardware and/or software suitably configured toprotect CMS components and/or enterprise computing resources from usersof other networks. Further, a firewall may be configured to limit orrestrict access to various systems and components behind the firewallfor web clients connecting through a web server. Firewall may reside invarying configurations including Stateful Inspection, Proxy based andPacket Filtering among others. Firewall may be integrated within an webserver or any other CMS components or may further reside as a separateentity.

The computers discussed herein may provide a suitable website or otherInternet-based graphical user interface which is accessible by users. Inone embodiment, the Microsoft Internet Information Server (IIS),Microsoft Transaction Server (MTS), and Microsoft SQL Server, are usedin conjunction with the Microsoft operating system, Microsoft NT webserver software, a Microsoft SQL Server database system, and a MicrosoftCommerce Server. Additionally, components such as Access or MicrosoftSQL Server, Oracle, Sybase, Informix MySQL, Interbase, etc., may be usedto provide an Active Data Object (ADO) compliant database managementsystem.

Any of the communications, inputs, storage, databases or displaysdiscussed herein may be facilitated through a website having web pages.The term “web page” as it is used herein is not meant to limit the typeof documents and applications that might be used to interact with theuser. For example, a typical website might include, in addition tostandard HTML documents, various forms, Java applets, JavaScript, activeserver pages (ASP), common gateway interface scripts (CGI), extensiblemarkup language (XML), dynamic HTML, cascading style sheets (CSS),helper applications, plug-ins, and the like. A server may include a webservice that receives a request from a web server, the request includinga URL (http://yahoo.com/stockquotes/ge) and an IP address(123.56.789.234). The web server retrieves the appropriate web pages andsends the data or applications for the web pages to the IP address. Webservices are applications that are capable of interacting with otherapplications over a communications means, such as the internet. Webservices are typically based on standards or protocols such as XML,XSLT, SOAP, WSDL and UDDI. Web services methods are well known in theart, and are covered in many standard texts. See, e.g., ALEX NGHIEM, ITWEB SERVICES: A ROADMAP FOR THE ENTERPRISE (2003), hereby incorporatedby reference.

The web-based clinical database for the system and method of the presentinvention preferably has the ability to upload and store clinical datafiles in native formats and is searchable on any clinical parameter. Thedatabase is also scalable and may use an EAV data model (metadata) toenter clinical annotations from any study for easy integration withother studies. In addition, the web-based clinical database is flexibleand may be XML and XSLT enabled to be able to add user customizedquestions dynamically. Further, the database includes exportability toCDISC ODM.

Practitioners will also appreciate that there are a number of methodsfor displaying data within a browser-based document. Data may berepresented as standard text or within a fixed list, scrollable list,drop-down list, editable text field, fixed text field, pop-up window,and the like. Likewise, there are a number of methods available formodifying data in a web page such as, for example, free text entry usinga keyboard, selection of menu items, check boxes, option boxes, and thelike.

The system and method may be described herein in terms of functionalblock components, screen shots, optional selections and variousprocessing steps. It should be appreciated that such functional blocksmay be realized by any number of hardware and/or software componentsconfigured to perform the specified functions. For example, the systemmay employ various integrated circuit components, e.g., memory elements,processing elements, logic elements, look-up tables, and the like, whichmay carry out a variety of functions under the control of one or moremicroprocessors or other control devices. Similarly, the softwareelements of the system may be implemented with any programming orscripting language such as C, C++, Macromedia Cold Fusion, MicrosoftActive Server Pages, Java, COBOL, assembler, PERL, Visual Basic, SQLStored Procedures, extensible markup language (XML), with the variousalgorithms being implemented with any combination of data structures,objects, processes, routines or other programming elements. Further, itshould be noted that the system may employ any number of conventionaltechniques for data transmission, signaling, data processing, networkcontrol, and the like. Still further, the system could be used to detector prevent security issues with a client-side scripting language, suchas JavaScript, VBScript or the like. For a basic introduction ofcryptography and network security, see any of the following references:(1) “Applied Cryptography: Protocols, Algorithms, And Source Code In C,”by Bruce Schneier, published by John Wiley & Sons (second edition,1995); (2) “Java Cryptography” by Jonathan Knudson, published byO'Reilly & Associates (1998); (3) “Cryptography & Network Security:Principles & Practice” by William Stallings, published by Prentice Hall;all of which are hereby incorporated by reference.

As used herein, the term “end user”, “consumer”, “customer”, “client”,“treating physician”, “hospital”, or “business” may be usedinterchangeably with each other, and each shall mean any person, entity,machine, hardware, software or business. Each participant is equippedwith a computing device in order to interact with the system andfacilitate online data access and data input. The customer has acomputing unit in the form of a personal computer, although other typesof computing units may be used including laptops, notebooks, hand heldcomputers, set-top boxes, cellular telephones, touch-tone telephones andthe like. The owner/operator of the system and method of the presentinvention has a computing unit implemented in the form of acomputer-server, although other implementations are contemplated by thesystem including a computing center shown as a main frame computer, amini-computer, a PC server, a network of computers located in the sameof different geographic locations, or the like. Moreover, the systemcontemplates the use, sale or distribution of any goods, services orinformation over any network having similar functionality describedherein.

In one illustrative embodiment, each client customer may be issued an“account” or “account number”. As used herein, the account or accountnumber may include any device, code, number, letter, symbol, digitalcertificate, smart chip, digital signal, analog signal, biometric orother identifier/indicia suitably configured to allow the consumer toaccess, interact with or communicate with the system (e.g., one or moreof an authorization/access code, personal identification number (PIN),Internet code, other identification code, and/or the like). The accountnumber may optionally be located on or associated with a charge card,credit card, debit card, prepaid card, embossed card, smart card,magnetic stripe card, bar code card, transponder, radio frequency cardor an associated account. The system may include or interface with anyof the foregoing cards or devices, or a fob having a transponder andRFID reader in RF communication with the fob. Although the system mayinclude a fob embodiment, the invention is not to be so limited. Indeed,system may include any device having a transponder which is configuredto communicate with RFID reader via RF communication. Typical devicesmay include, for example, a key ring, tag, card, cell phone, wristwatchor any such form capable of being presented for interrogation. Moreover,the system, computing unit or device discussed herein may include a“pervasive computing device,” which may include a traditionallynon-computerized device that is embedded with a computing unit. Theaccount number may be distributed and stored in any form of plastic,electronic, magnetic, radio frequency, wireless, audio and/or opticaldevice capable of transmitting or downloading data from itself to asecond device.

As will be appreciated by one of ordinary skill in the art, the systemmay be embodied as a customization of an existing system, an add-onproduct, upgraded software, a standalone system, a distributed system, amethod, a data processing system, a device for data processing, and/or acomputer program product. Accordingly, the system may take the form ofan entirely software embodiment, an entirely hardware embodiment, or anembodiment combining aspects of both software and hardware. Furthermore,the system may take the form of a computer program product on acomputer-readable storage medium having computer-readable program codemeans embodied in the storage medium. Any suitable computer-readablestorage medium may be used, including hard disks, CD-ROM, opticalstorage devices, magnetic storage devices, and/or the like.

The system and method is described herein with reference to screenshots, block diagrams and flowchart illustrations of methods, apparatus(e.g., systems), and computer program products according to variousembodiments. It will be understood that each functional block of theblock diagrams and the flowchart illustrations, and combinations offunctional blocks in the block diagrams and flowchart illustrations,respectively, can be implemented by computer program instructions.

These computer program instructions may be loaded onto a general purposecomputer, special purpose computer, or other programmable dataprocessing apparatus to produce a machine, such that the instructionsthat execute on the computer or other programmable data processingapparatus create means for implementing the functions specified in theflowchart block or blocks. These computer program instructions may alsobe stored in a computer-readable memory that can direct a computer orother programmable data processing apparatus to function in a particularmanner, such that the instructions stored in the computer-readablememory produce an article of manufacture including instruction meanswhich implement the function specified in the flowchart block or blocks.The computer program instructions may also be loaded onto a computer orother programmable data processing apparatus to cause a series ofoperational steps to be performed on the computer or other programmableapparatus to produce a computer-implemented process such that theinstructions which execute on the computer or other programmableapparatus provide steps for implementing the functions specified in theflowchart block or blocks.

Accordingly, functional blocks of the block diagrams and flowchartillustrations support combinations of means for performing the specifiedfunctions, combinations of steps for performing the specified functions,and program instruction means for performing the specified functions. Itwill also be understood that each functional block of the block diagramsand flowchart illustrations, and combinations of functional blocks inthe block diagrams and flowchart illustrations, can be implemented byeither special purpose hardware-based computer systems which perform thespecified functions or steps, or suitable combinations of specialpurpose hardware and computer instructions. Further, illustrations ofthe process flows and the descriptions thereof may make reference touser windows, web pages, websites, web forms, prompts, etc.Practitioners will appreciate that the illustrated steps describedherein may comprise in any number of configurations including the use ofwindows, web pages, web forms, popup windows, prompts and the like. Itshould be further appreciated that the multiple steps as illustrated anddescribed may be combined into single web pages and/or windows but havebeen expanded for the sake of simplicity. In other cases, stepsillustrated and described as single process steps may be separated intomultiple web pages and/or windows but have been combined for simplicity.

Molecular Profiling Methods

FIG. 1 illustrates a block diagram of an illustrative embodiment of asystem 10 for determining individualized medical intervention for aparticular disease state that uses molecular profiling of a patient'sbiological specimen. System 10 includes a user interface 12, a hostserver 14 including a processor 16 for processing data, a memory 18coupled to the processor, an application program 20 stored in the memory18 and accessible by the processor 16 for directing processing of thedata by the processor 16, a plurality of internal databases 22 andexternal databases 24, and an interface with a wired or wirelesscommunications network 26 (such as the Internet, for example). System 10may also include an input digitizer 28 coupled to the processor 16 forinputting digital data from data that is received from user interface12.

User interface 12 includes an input device 30 and a display 32 forinputting data into system 10 and for displaying information derivedfrom the data processed by processor 16. User interface 12 may alsoinclude a printer 34 for printing the information derived from the dataprocessed by the processor 16 such as patient reports that may includetest results for targets and proposed drug therapies based on the testresults.

Internal databases 22 may include, but are not limited to, patientbiological sample/specimen information and tracking, clinical data,patient data, patient tracking, file management, study protocols,patient test results from molecular profiling, and billing informationand tracking. External databases 24 nay include, but are not limited to,drug libraries, gene libraries, disease libraries, and public andprivate databases such as UniGene, OMIM, GO, TIGR, GenBank, KEGG andBiocarta.

Various methods may be used in accordance with system 10. FIG. 2 shows aflowchart of an illustrative embodiment of a method 50 for determiningindividualized medical intervention for a particular disease state thatuses molecular profiling of a patient's biological specimen that is nondisease specific. In order to determine a medical intervention for aparticular disease state using molecular profiling that is independentof disease lineage diagnosis (i.e. not single disease restricted), atleast one test is performed for at least one target from a biologicalsample of a diseased patient in step 52. A target is defined as anymolecular finding that may be obtained from molecular testing. Forexample, a target may include one or more genes, one or more geneexpressed proteins, one or more molecular mechanisms, and/orcombinations of such. For example, the expression level of a target canbe determined by the analysis of mRNA levels or the target or gene, orprotein levels of the gene. Tests for finding such targets may include,but are not limited, fluorescent in-situ hybridization (FISH), in-situhybridization (ISH), and other molecular tests known to those skilled inthe art. PCR-based methods, such as real-time PCR or quantitative PCRcan be used. Furthermore, microarray analysis, such as a comparativegenomic hybridization (CGH) micro array, a single nucleotidepolymorphism (SNP) microarray, a proteomic array, or antibody arrayanalysis can also be used in the methods disclosed herein. In someembodiments, microarray analysis comprises identifying whether a gene isup-regulated or down-regulated relative to a reference with asignificance of p<0.001. Tests or analyses of targets can also compriseimmunohistochemical (IHC) analysis. In some embodiments, IHC analysiscomprises determining whether 30% or more of a sample is stained, if thestaining intensity is +2 or greater, or both.

Furthermore, the methods disclosed herein also including profiling morethan one target. For example, the expression of a plurality of genes canbe identified. Furthermore, identification of a plurality of targets ina sample can be by one method or by various means. For example, theexpression of a first gene can be determined by one method and theexpression level of a second gene determined by a different method.Alternatively, the same method can be used to detect the expressionlevel of the first and second gene. For example, the first method can beIHC and the second by microarray analysis, such as detecting the geneexpression of a gene.

In some embodiments, molecular profiling can also including identifyinga genetic variant, such as a mutation, polymorphism (such as a SNP),deletion, or insertion of a target. For example, identifying a SNP in agene can be determined by microarray analysis, real-time PCR, orsequencing. Other methods disclosed herein can also be used to identifyvariants of one or more targets.

Accordingly, one or more of the following may be performed: an IHCanalysis in step 54, a microanalysis in step 56, and other moleculartests know to those skilled in the art in step 58.

Biological samples are obtained from diseased patients by taking abiopsy of a tumor, conducting minimally invasive surgery if no recenttumor is available, obtaining a sample of the patient's blood, or asample of any other biological fluid including, but not limited to, cellextracts, nuclear extracts, cell lysates or biological products orsubstances of biological origin such as excretions, blood, sera, plasma,urine, sputum, tears, feces, saliva, membrane extracts, and the like.

In step 60, a determination is made as to whether one or more of thetargets that were tested for in step 52 exhibit a change in expressioncompared to a normal reference for that particular target. In oneillustrative method of the invention, an IHC analysis may be performedin step 54 and a determination as to whether any targets from the IHCanalysis exhibit a change in expression is made in step 64 bydetermining whether 30% or more of the biological sample cells were +2or greater staining for the particular target. It will be understood bythose skilled in the art that there will be instances where +1 orgreater staining will indicate a change in expression in that stainingresults may vary depending on the technician performing the test andtype of target being tested. In another illustrative embodiment of theinvention, a micro array analysis may be performed in step 56 and adetermination as to whether any targets from the micro array analysisexhibit a change in expression is made in step 66 by identifying whichtargets are up-regulated or down-regulated by determining whether thefold change in expression for a particular target relative to a normaltissue of origin reference is significant at p<0.001. A change inexpression may also be evidenced by an absence of one or more genes,gene expressed proteins, molecular mechanisms, or other molecularfindings.

After determining which targets exhibit a change in expression in step60, at least one non-disease specific agent is identified that interactswith each target having a changed expression in step 70. An agent may beany drug or compound having a therapeutic effect. A non-disease specificagent is a therapeutic drug or compound not previously associated withtreating the patient's diagnosed disease that is capable of interactingwith the target from the patient's biological sample that has exhibiteda change in expression. Some of the non-disease specific agents thathave been found to interact with specific targets found in differentcancer patients are shown in Table 5 below.

TABLE 5 Illustrative target-drug associations Patients Target(s) FoundTreatment(s) Advanced Pancreatic Cancer HER 2/neu Trastuzumab AdvancedPancreatic Cancer EGFR, HIF 1α Cetuximab, Sirolimus Advanced OvarianCancer ERCC3 Irofulven Advanced Adenoid Cystic Vitamin D receptors,Calcitriol, Carcinoma Androgen receptors Flutamide

Finally, in step 80, a patient profile report may be provided whichincludes the patient's test results for various targets and any proposedtherapies based on those results. An illustrative patient profile report100 is shown in FIGS. 3A-3D. Patient profile report 100 shown in FIG. 3Aidentifies the targets tested 102, those targets tested that exhibitedsignificant changes in expression 104, and proposed non-disease specificagents for interacting with the targets 106. Patient profile report 100shown in FIG. 3B identifies the results 108 of immunohistochemicalanalysis for certain gene expressed proteins 110 and whether a geneexpressed protein is a molecular target 112 by determining whether 30%or more of the tumor cells were +2 or greater staining. Report 100 alsoidentifies immunohistochemical tests that were not performed 114.Patient profile report 100 shown in FIG. 3C identifies the genesanalyzed 116 with a micro array analysis and whether the genes wereunder expressed or over expressed 118 compared to a reference. Finally,patient profile report 100 shown in FIG. 3D identifies the clinicalhistory 120 of the patient and the specimens that were submitted 122from the patient. Molecular profiling techniques can be performedanywhere, e.g., a foreign country, and the results sent by network to anappropriate party, e.g., the patient, a physician, lab or other partylocated remotely.

FIG. 4 shows a flowchart of an illustrative embodiment of a method 200for identifying a drug therapy/agent capable of interacting with atarget. In step 202, a molecular target is identified which exhibits achange in expression in a number of diseased individuals. Next, in step204, a drug therapy/agent is administered to the diseased individuals.After drug therapy/agent administration, any changes in the moleculartarget identified in step 202 are identified in step 206 in order todetermine if the drug therapy/agent administered in step 204 interactswith the molecular targets identified in step 202. If it is determinedthat the drug therapy/agent administered in step 204 interacts with amolecular target identified in step 202, the drug therapy/agent may beapproved for treating patients exhibiting a change in expression of theidentified molecular target instead of approving the drug therapy/agentfor a particular disease.

FIGS. 5-14 are flowcharts and diagrams illustrating various parts of aninformation-based personalized medicine drug discovery system and methodin accordance with the present invention. FIG. 5 is a diagram showing anillustrative clinical decision support system of the information-basedpersonalized medicine drug discovery system and method of the presentinvention. Data obtained through clinical research and clinical caresuch as clinical trial data, biomedical/molecular imaging data,genomics/proteomics/chemical library/literature/expert curation,biospecimen tracking/LIMS, family history/environmental records, andclinical data are collected and stored as databases and datamarts withina data warehouse. FIG. 6 is a diagram showing the flow of informationthrough the clinical decision support system of the information-basedpersonalized medicine drug discovery system and method of the presentinvention using web services. A user interacts with the system byentering data into the system via form-based entry/upload of data sets,formulating queries and executing data analysis jobs, and acquiring andevaluating representations of output data. The data warehouse in the webbased system is where data is extracted, transformed, and loaded fromvarious database systems. The data warehouse is also where commonformats, mapping and transformation occurs. The web based system alsoincludes datamarts which are created based on data views of interest.

flow chart of an illustrative clinical decision support system of theinformation-based personalized medicine drug discovery system and methodof the present invention is shown in FIG. 7. The clinical informationmanagement system includes the laboratory information management systemand the medical information contained in the data warehouses anddatabases includes medical information libraries, such as druglibraries, gene libraries, and disease libraries, in addition toliterature text mining. Both the information management systems relatingto particular patients and the medical information databases and datawarehouses come together at a data junction center where diagnosticinformation and therapeutic options can be obtained. A financialmanagement system may also be incorporated in the clinical decisionsupport system of the information-based personalized medicine drugdiscovery system and method of the present invention.

FIG. 8 is a diagram showing an illustrative biospecimen tracking andmanagement system which may be used as part of the information-basedpersonalized medicine drug discovery system and method of the presentinvention. FIG. 8 shows two host medical centers which forward specimensto a tissue/blood bank. The specimens may go through laboratory analysisprior to shipment. Research may also be conducted on the samples viamicro array, genotyping, and proteomic analysis. This information can beredistributed to the tissue/blood bank. FIG. 9 depicts a flow chart ofan illustrative biospecimen tracking and management system which may beused with the information-based personalized medicine drug discoverysystem and method of the present invention. The host medical centerobtains samples from patients and then ships the patient samples to amolecular profiling laboratory which may also perform RNA and DNAisolation and analysis.

A diagram showing a method for maintaining a clinical standardizedvocabulary for use with the information-based personalized medicine drugdiscovery system and method of the present invention is shown in FIG.10. FIG. 10 illustrates how physician observations and patientinformation associated with one physician's patient may be madeaccessible to another physician to enable the other physician to use thedata in making diagnostic and therapeutic decisions for their patients.

FIG. 11 shows a schematic of an illustrative microarray gene expressiondatabase which may be used as part of the information-based personalizedmedicine drug discovery system and method of the present invention. Themicro array gene expression database includes both external databasesand internal databases which can be accessed via the web based system.External databases may include, but are not limited to, UniGene, GO,TIGR, GenBank, KEGG. The internal databases may include, but are notlimited to, tissue tracking, LIMS, clinical data, and patient tracking.FIG. 12 shows a diagram of an illustrative micro array gene expressiondatabase data warehouse which may be used as part of theinformation-based personalized medicine drug discovery system and methodof the present invention. Laboratory data, clinical data, and patientdata may all be housed in the micro array gene expression database datawarehouse and the data may in turn be accessed by public/private releaseand used by data analysis tools.

Another schematic showing the flow of information through aninformation-based personalized medicine drug discovery system and methodof the present invention is shown in FIG. 13. Like FIG. 7, the schematicincludes clinical information management, medical and literatureinformation management, and financial management of theinformation-based personalized medicine drug discovery system and methodof the present invention. FIG. 14 is a schematic showing an illustrativenetwork of the information-based personalized medicine drug discoverysystem and method of the present invention. Patients, medicalpractitioners, host medical centers, and labs all share and exchange avariety of information in order to provide a patient with a proposedtherapy or agent based on various identified targets.

FIGS. 15-25 are computer screen print outs associated with various partsof the information-based personalized medicine drug discovery system andmethod shown in FIGS. 5-14. FIGS. 15 and 16 show computer screens wherephysician information and insurance company information is entered onbehalf of a client. FIGS. 17-19 show computer screens in whichinformation can be entered for ordering analysis and tests on patientsamples.

FIG. 20 is a computer screen showing micro array analysis results ofspecific genes tested with patient samples. This information andcomputer screen is similar to the information detailed in the patientprofile report shown in FIG. 3C. FIG. 22 is a computer screen that showsimmunohistochemistry test results for a particular patient for variousgenes. This information is similar to the information contained in thepatient profile report shown in FIG. 3B.

FIG. 21 is a computer screen showing selection options for findingparticular patients, ordering tests and/or results, issuing patientreports, and tracking current cases/patients.

FIG. 23 is a computer screen which outlines some of the steps forcreating a patient profile report as shown in FIGS. 3A through 3D. FIG.24 shows a computer screen for ordering an immunohistochemistry test ona patient sample and FIG. 25 shows a computer screen for enteringinformation regarding a primary tumor site for micro array analysis. Itwill be understood by those skilled in the art that any number andvariety of computer screens may be used to enter the informationnecessary for using the information-based personalized medicine drugdiscovery system and method of the present invention and to obtaininformation resulting from using the information-based personalizedmedicine drug discovery system and method of the present invention.

The systems of the invention can be used to automate the steps ofidentifying a molecular profile to assess a cancer. In an aspect, theinvention provides a method of generating a report comprising amolecular profile. The method comprises: performing a search on anelectronic medium to obtain a data set, wherein the data set comprises aplurality of scientific publications corresponding to plurality ofcancer biomarkers; and analyzing the data set to identify a rule setlinking a characteristic of each of the plurality of cancer biomarkerswith an expected benefit of a plurality of treatment options, therebyidentifying the cancer biomarkers included within a molecular profile.The method can further comprise performing molecular profiling on asample from a subject to assess the characteristic of each of theplurality of cancer biomarkers, and compiling a report comprising theassessed characteristics into a list, thereby generating a report thatidentifies a molecular profile for the sample. The report can furthercomprise a list describing the expected benefit of the plurality oftreatment options based on the assessed characteristics, therebyidentifying candidate treatment options for the subject. The sample fromthe subject may comprise cancer cells. The cancer can be any cancerdisclosed herein or known in the art.

The characteristic of each of the plurality of cancer biomarkers can beany useful characteristic for molecular profiling as disclosed herein orknown in the art. Such characteristics include without limitationmutations (point mutations, insertions, deletions, rearrangements, etc),epigenetic modifications, copy number, nucleic acid or proteinexpression levels, post-translational modifications, and the like.

In an embodiment, the method further comprises identifying a prioritylist as amongst said plurality of cancer biomarkers. The priority listcan be sorted according to any appropriate priority criteria. In anembodiment, the priority list is sorted according to strength ofevidence in the plurality of scientific publications linking the cancerbiomarkers to the expected benefit. In another embodiment, the prioritylist is sorted according to strength of the expected benefit. In stillanother embodiment, the priority list is sorted according to strength ofthe expected benefit. One of skill will appreciate that the prioritylist can be sorted according to a combination of these or otherappropriate priority criteria. The candidate treatment options can besorted according to the priority list, thereby identifying a ranked listof treatment options for the subject.

The candidate treatment options can be categorized by expected benefitto the subject. For example, the candidate treatment options cancategorized as those that are expected to provide benefit, those thatare not expected to provide benefit, or those whose expected benefitcannot be determined.

The candidate treatment options can include regulatory approved and/oron-compendium treatments for the cancer. The candidate treatment optionscan include regulatory approved but off-label treatments for the cancer,such as a treatment that has been approved for a cancer of anotherlineage. The candidate treatment options can include treatments that areunder development, such as in ongoing clinical trials. The report mayidentify treatments as approved, on- or off-compendium, in clinicaltrials, and the like.

In some embodiments, the method further comprises analyzing the data setto select a laboratory technique to assess the characteristics of thebiomarkers, thereby designating a technique that can be used to assessthe characteristic for each of the plurality of biomarkers. In otherembodiments, the laboratory technique is chosen based on itsapplicability to assess the characteristic of each of the biomarkers.The laboratory techniques can be those disclosed herein, includingwithout limitation FISH for gene copy number or mutation analysis, IHCfor protein expression levels, RT-PCR for mutation or expressionanalysis, sequencing or fragment analysis for mutation analysis.Sequencing includes any useful sequencing method disclosed herein orknown in the art, including without limitation Sanger sequencing,pyrosequencing, or next generation sequencing methods.

In a related aspect, the invention provides a method comprising:performing a search on an electronic medium to obtain a data setcomprising a plurality of scientific publications corresponding toplurality of cancer biomarkers; analyzing the data set to select amethod to assess a characteristic of each of the cancer biomarkers,thereby designating a method for characterizing each of the biomarkers;further analyzing the data set to select a rule set that identifies apriority list as amongst the biomarkers; performing tumor profiling on atumor sample from a subject comprising the selected methods to determinethe status of the characteristic of each of the biomarkers; andcompiling the status in a report according to said priority list;thereby generating a report that identifies a tumor profile.

Molecular Profiling Targets

The present invention provides methods and systems for analyzingdiseased tissue using molecular profiling as previously described above.Because the methods rely on analysis of the characteristics of the tumorunder analysis, the methods can be applied in for any tumor or any stageof disease, such an advanced stage of disease or a metastatic tumor ofunknown origin. As described herein, a tumor or cancer sample isanalyzed for molecular characteristics in order to predict or identify acandidate therapeutic treatment. The molecular characteristics caninclude the expression of genes or gene products, assessment of genecopy number, or mutational analysis. Any relevant determinablecharacteristic that can assist in prediction or identification of acandidate therapeutic can be included within the methods of theinvention.

The biomarker patterns or biomarker signature sets can be determined fortumor types, diseased tissue types, or diseased cells including withoutlimitation adipose, adrenal cortex, adrenal gland, adrenalgland—medulla, appendix, bladder, blood vessel, bone, bone cartilage,brain, breast, cartilage, cervix, colon, colon sigmoid, dendritic cells,skeletal muscle, endometrium, esophagus, fallopian tube, fibroblast,gallbladder, kidney, larynx, liver, lung, lymph node, melanocytes,mesothelial lining, myoepithelial cells, osteoblasts, ovary, pancreas,parotid, prostate, salivary gland, sinus tissue, skeletal muscle, skin,small intestine, smooth muscle, stomach, synovium, joint lining tissue,tendon, testis, thymus, thyroid, uterus, and uterus corpus.

The methods of the present invention can be used for selecting atreatment of any cancer or tumor type, including but not limited tobreast cancer (including HER2+ breast cancer, HER2− breast cancer,ER/PR+, HER2− breast cancer, or triple negative breast cancer),pancreatic cancer, cancer of the colon and/or rectum, leukemia, skincancer, bone cancer, prostate cancer, liver cancer, lung cancer, braincancer, cancer of the larynx, gallbladder, parathyroid, thyroid,adrenal, neural tissue, head and neck, stomach, bronchi, kidneys, basalcell carcinoma, squamous cell carcinoma of both ulcerating and papillarytype, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma,veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lungtumor, islet cell carcinoma, primary brain tumor, acute and chroniclymphocytic and granulocytic tumors, hairy-cell tumor, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuroma,intestinal ganglioneuroma, hyperplastic corneal nerve tumor, marfanoidhabitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyoma,cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma,soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosisfungoides, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and othersarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera,adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignantmelanomas, and epidermoid carcinomas. The cancer or tumor can comprise,without limitation, a carcinoma, a sarcoma, a lymphoma or leukemia, agerm cell tumor, a blastoma, or other cancers. Carcinomas that can beassessed using the subject methods include without limitation epithelialneoplasms, squamous cell neoplasms, squamous cell carcinoma, basal cellneoplasms basal cell carcinoma, transitional cell papillomas andcarcinomas, adenomas and adenocarcinomas (glands), adenoma,adenocarcinoma, linitis plastica insulinoma, glucagonoma, gastrinoma,vipoma, cholangiocarcinoma, hepatocellular carcinoma, adenoid cysticcarcinoma, carcinoid tumor of appendix, prolactinoma, oncocytoma,hurthle cell adenoma, renal cell carcinoma, grawitz tumor, multipleendocrine adenomas, endometrioid adenoma, adnexal and skin appendageneoplasms, mucoepidermoid neoplasms, cystic, mucinous and serousneoplasms, cystadenoma, pseudomyxoma peritonei, ductal, lobular andmedullary neoplasms, acinar cell neoplasms, complex epithelialneoplasms, warthin's tumor, thymoma, specialized gonadal neoplasms, sexcord stromal tumor, thecoma, granulosa cell tumor, arrhenoblastoma,sertoli leydig cell tumor, glomus tumors, paraganglioma,pheochromocytoma, glomus tumor, nevi and melanomas, melanocytic nevus,malignant melanoma, melanoma, nodular melanoma, dysplastic nevus,lentigo maligna melanoma, superficial spreading melanoma, and malignantacral lentiginous melanoma. Sarcoma that can be assessed using thesubject methods include without limitation Askin's tumor, botryodies,chondrosarcoma, Ewing's sarcoma, malignant hemangio endothelioma,malignant schwannoma, osteosarcoma, soft tissue sarcomas including:alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes,dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor,epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletalosteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma,kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma,lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma,rhabdomyosarcoma, and synovialsarcoma. Lymphoma and leukemia that can beassessed using the subject methods include without limitation chroniclymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocyticleukemia, lymphoplasmacytic lymphoma (such as waldenstrommacroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma,plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chaindiseases, extranodal marginal zone B cell lymphoma, also called maltlymphoma, nodal marginal zone B cell lymphoma (nmzl), follicularlymphoma, mantle cell lymphoma, diffuse large B cell lymphoma,mediastinal (thymic) large B cell lymphoma, intravascular large B celllymphoma, primary effusion lymphoma, burkitt lymphoma/leukemia, T cellprolymphocytic leukemia, T cell large granular lymphocytic leukemia,aggressive NK cell leukemia, adult T cell leukemia/lymphoma, extranodalNK/T cell lymphoma, nasal type, enteropathy-type T cell lymphoma,hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosisfungoides/sezary syndrome, primary cutaneous CD30-positive T celllymphoproliferative disorders, primary cutaneous anaplastic large celllymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma,peripheral T cell lymphoma, unspecified, anaplastic large cell lymphoma,classical Hodgkin lymphomas (nodular sclerosis, mixed cellularity,lymphocyte-rich, lymphocyte depleted or not depleted), and nodularlymphocyte-predominant Hodgkin lymphoma. Germ cell tumors that can beassessed using the subject methods include without limitation germinoma,dysgerminoma, seminoma, nongerminomatous germ cell tumor, embryonalcarcinoma, endodermal sinus turmor, choriocarcinoma, teratoma,polyembryoma, and gonadoblastoma. Blastoma includes without limitationnephroblastoma, medulloblastoma, and retinoblastoma. Other cancersinclude without limitation labial carcinoma, larynx carcinoma,hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma,gastric carcinoma, adenocarcinoma, thyroid cancer (medullary andpapillary thyroid carcinoma), renal carcinoma, kidney parenchymacarcinoma, cervix carcinoma, uterine corpus carcinoma, endometriumcarcinoma, chorion carcinoma, testis carcinoma, urinary carcinoma,melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma,medulloblastoma and peripheral neuroectodermal tumors, gall bladdercarcinoma, bronchial carcinoma, multiple myeloma, basalioma, teratoma,retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma,craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma,liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmocytoma.

In an embodiment, the cancer may be a acute myeloid leukemia (AML),breast carcinoma, cholangiocarcinoma, colorectal adenocarcinoma,extrahepatic bile duct adenocarcinoma, female genital tract malignancy,gastric adenocarcinoma, gastroesophageal adenocarcinoma,gastrointestinal stromal tumors (GIST), glioblastoma, head and necksquamous carcinoma, leukemia, liver hepatocellular carcinoma, low gradeglioma, lung bronchioloalveolar carcinoma (BAC), lung non-small celllung cancer (NSCLC), lung small cell cancer (SCLC), lymphoma, malegenital tract malignancy, malignant solitary fibrous tumor of the pleura(MSFT), melanoma, multiple myeloma, neuroendocrine tumor, nodal diffuselarge B-cell lymphoma, non epithelial ovarian cancer (non-EOC), ovariansurface epithelial carcinoma, pancreatic adenocarcinoma, pituitarycarcinomas, oligodendroglioma, prostatic adenocarcinoma, retroperitonealor peritoneal carcinoma, retroperitoneal or peritoneal sarcoma, smallintestinal malignancy, soft tissue tumor, thymic carcinoma, thyroidcarcinoma, or uveal melanoma.

In a further embodiment, the cancer may be a lung cancer includingnon-small cell lung cancer and small cell lung cancer (including smallcell carcinoma (oat cell cancer), mixed small cell/large cell carcinoma,and combined small cell carcinoma), colon cancer, breast cancer,prostate cancer, liver cancer, pancreas cancer, brain cancer, kidneycancer, ovarian cancer, stomach cancer, skin cancer, bone cancer,gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma,hepatocellular carcinoma, papillary renal carcinoma, head and necksquamous cell carcinoma, leukemia, lymphoma, myeloma, or a solid tumor.

In embodiments, the cancer comprises an acute lymphoblastic leukemia;acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancers;AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas;atypical teratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer;brain stem glioma; brain tumor (including brain stem glioma, centralnervous system atypical teratoid/rhabdoid tumor, central nervous systemembryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma,ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymaltumors of intermediate differentiation, supratentorial primitiveneuroectodermal tumors and pineoblastoma); breast cancer; bronchialtumors; Burkitt lymphoma; cancer of unknown primary site; carcinoidtumor; carcinoma of unknown primary site; central nervous systematypical teratoid/rhabdoid tumor; central nervous system embryonaltumors; cervical cancer; childhood cancers; chordoma; chroniclymphocytic leukemia; chronic myelogenous leukemia; chronicmyeloproliferative disorders; colon cancer; colorectal cancer;craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas isletcell tumors; endometrial cancer; ependymoblastoma; ependymoma;esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranialgerm cell tumor; extragonadal germ cell tumor; extrahepatic bile ductcancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinalcarcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinalstromal tumor (GIST); gestational trophoblastic tumor; glioma; hairycell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma;hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposisarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer;lip cancer; liver cancer; malignant fibrous histiocytoma bone cancer;medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma;Merkel cell skin carcinoma; mesothelioma; metastatic squamous neckcancer with occult primary; micropapillary urothelial carcinoma; mouthcancer; multiple endocrine neoplasia syndromes; multiple myeloma;multiple myeloma/plasma cell neoplasm; mycosis fungoides;myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavitycancer; nasopharyngeal cancer; neuroblastoma; Non-Hodgkin lymphoma;nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oralcavity cancer; oropharyngeal cancer; osteosarcoma; other brain andspinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovariangerm cell tumor; ovarian low malignant potential tumor; pancreaticcancer; papillomatosis; paranasal sinus cancer; parathyroid cancer;pelvic cancer; penile cancer; pharyngeal cancer; pineal parenchymaltumors of intermediate differentiation; pineoblastoma; pituitary tumor;plasma cell neoplasm/multiple myeloma; pleuropulmonary blastoma; primarycentral nervous system (CNS) lymphoma; primary hepatocellular livercancer; prostate cancer; rectal cancer; renal cancer; renal cell(kidney) cancer; renal cell cancer; respiratory tract cancer;retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sézarysyndrome; small cell lung cancer; small intestine cancer; soft tissuesarcoma; squamous cell carcinoma; squamous neck cancer; stomach(gastric) cancer; supratentorial primitive neuroectodermal tumors;T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma;thymoma; thyroid cancer; transitional cell cancer; transitional cellcancer of the renal pelvis and ureter; trophoblastic tumor; uretercancer; urethral cancer; uterine cancer; uterine sarcoma; vaginalcancer; vulvar cancer; Waldenstrom macroglobulinemia; or Wilm's tumor.

The methods of the invention can be used to determine biomarker patternsor biomarker signature sets in a number of tumor types, diseased tissuetypes, or diseased cells including accessory, sinuses, middle and innerear, adrenal glands, appendix, hematopoietic system, bones and joints,spinal cord, breast, cerebellum, cervix uteri, connective and softtissue, corpus uteri, esophagus, eye, nose, eyeball, fallopian tube,extrahepatic bile ducts, other mouth, intrahepatic bile ducts, kidney,appendix-colon, larynx, lip, liver, lung and bronchus, lymph nodes,cerebral, spinal, nasal cartilage, excl. retina, eye, nos, oropharynx,other endocrine glands, other female genital, ovary, pancreas, penis andscrotum, pituitary gland, pleura, prostate gland, rectum renal pelvis,ureter, peritonem, salivary gland, skin, small intestine, stomach,testis, thymus, thyroid gland, tongue, unknown, urinary bladder, uterus,nos, vagina & labia, and vulva, nos.

In some embodiments, the molecular profiling methods are used toidentify a treatment for a cancer of unknown primary (CUP).Approximately 40,000 CUP cases are reported annually in the US. Most ofthese are metastatic and/or poorly differentiated tumors. Becausemolecular profiling can identify a candidate treatment depending onlyupon the diseased sample, the methods of the invention can be used inthe CUP setting. Moreover, molecular profiling can be used to createsignatures of known tumors, which can then be used to classify a CUP andidentify its origin. In an aspect, the invention provides a method ofidentifying the origin of a CUP, the method comprising performingmolecular profiling on a panel of diseased samples to determine a panelof molecular profiles that correlate with the origin of each diseasedsample, performing molecular profiling on a CUP sample, and correlatingthe molecular profile of the CUP sample with the molecular profiling ofthe panel of diseased samples, thereby identifying the origin of the CUPsample. The identification of the origin of the CUP sample can be madeby matching the molecular profile of the CUP sample with the molecularprofiles that correlate most closely from the panel of disease samples.The molecular profiling can use any of the techniques described herein,e.g., IHC, FISH, microarray and sequencing. The diseased samples and CUPsamples can be derived from a patient sample, e.g., a biopsy sample,including a fine needle biopsy. In one embodiment, DNA microarray andIHC profiling are performed on the panel of diseased samples, DNAmicroarray is performed on the CUP samples, and then IHC is performed onthe CUP sample for a subset of the most informative genes as indicatedby the DNA microarray analysis. This approach can identify the origin ofthe CUP sample while avoiding the expense of performing unnecessary IHCtesting. The IHC can be used to confirm the microarray findings.

The biomarker patterns or biomarker signature sets of the cancer ortumor can be used to determine a therapeutic agent or therapeuticprotocol that is capable of interacting with the biomarker pattern orsignature set. For example, with advanced breast cancer,immunohistochemistry analysis can be used to determine one or more geneexpressed proteins that are overexpressed. Accordingly, a biomarkerpattern or biomarker signature set can be identified for advanced stagebreast cancer and a therapeutic agent or therapeutic protocol can beidentified which is capable of interacting with the biomarker pattern orsignature set.

The biomarker patterns and/or biomarker signature sets can comprise atleast one biomarker. In yet other embodiments, the biomarker patterns orsignature sets can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10biomarkers. In some embodiments, the biomarker signature sets orbiomarker patterns can comprise at least 15, 20, 30, 40, 50, or 60biomarkers. In some embodiments, the biomarker signature sets orbiomarker patterns can comprise at least 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000 or50,000 biomarkers. Analysis of the one or more biomarkers can be by oneor more methods. For example, analysis of 2 biomarkers can be performedusing microarrays. Alternatively, one biomarker may be analyzed by IHCand another by microarray. Any such combinations of methods andbiomarkers are contemplated herein.

The one or more biomarkers can be selected from the group consisting of,but not limited to: Her2/Neu, ER, PR, c-kit, EGFR, MLH1, MSH2, CD20,p53, Cyclin D1, bc12, COX-2, Androgen receptor, CD52, PDGFR, AR, CD25,VEGF, HSP90, PTEN, RRM1, SPARC, Survivin, TOP2A, BCL2, HIF1A, AR, ESR1,PDGFRA, KIT, PDGFRB, CDW52, ZAP70, PGR, SPARC, GART, GSTP1, NFKBIA,MSH2, TXNRD1, HDAC1, PDGFC, PTEN, CD33, TYMS, RXRB, ADA, TNF, ERCC3,RAF1, VEGF, TOP1, TOP2A, BRCA2, TK1, FOLR2, TOP2B, MLH1, IL2RA, DNMT1,HSPCA, ERBR2, ERBB2, SSTR1, VHL, VDR, PTGS2, POLA, CES2, EGFR, OGFR,ASNS, NFKB2, RARA, MS4A1, DCK, DNMT3A, EREG, Epiregulin, FOLR1, GNRH1,GNRHR1, FSHB, FSHR, FSHPRH1, folate receptor, HGF, HIG1, IL13RA1, LTB,ODC1, PPARG, PPARGC1, Lymphotoxin Beta Receptor, Myc, Topoisomerase II,TOPO2B, TXN, VEGFC, ACE2, ADH1C, ADH4, AGT, AREG, CA2, CDK2, caveolin,NFKB1, ASNS, BDCA1, CD52, DHFR, DNMT3B, EPHA2, FLT1, HSP9OAA1, KDR, LCK,MGMT, RRM1, RRM2, RRM2B, RXRG, SRC, SSTR2, SSTR3, SSTR4, SSTR5, VEGFA,or YES1.

For example, a biological sample from an individual can be analyzed todetermine a biomarker pattern or biomarker signature set that comprisesa biomarker such as HSP90, Survivin, RRM1, SSTR53, DNMT3B, VEGFA, SSTR4,RRM2, SRC, RRM2B, HSP9OAA1, STR2, FLT1, SSTR5, YES1, BRCA1, RRM1, DHFR,KDR, EPHA2, RXRG, or LCK. In other embodiments, the biomarker SPARC,HSP90, TOP2A, PTEN, Survivin, or RRM1 forms part of the biomarkerpattern or biomarker signature set. In yet other embodiments, thebiomarker MGMT, SSTR53, DNMT3B, VEGFA, SSTR4, RRM2, SRC, RRM2B,HSP9OAA1, STR2, FLT1, SSTR5, YES1, BRCA1, RRM1, DHFR, KDR, EPHA2, RXRG,CD52, or LCK is included in a biomarker pattern or biomarker signatureset. In still other embodiments, the biomarker hENT1, cMet, P21, PARP-1,TLE3 or IGF1R is included in a biomarker pattern or biomarker signatureset.

The expression level of HSP90, Survivin, RRM1, SSTRS3, DNMT3B, VEGFA,SSTR4, RRM2, SRC, RRM2B, HSP9OAA1, STR2, FLT1, SSTR5, YES1, BRCA1, RRM1,DHFR, KDR, EPHA2, RXRG, or LCK can be determined and used to identify atherapeutic for an individual. The expression level of the biomarker canbe used to form a biomarker pattern or biomarker signature set.Determining the expression level can be by analyzing the levels of mRNAor protein, such as by microarray analysis or IHC. In some embodiments,the expression level of a biomarker is performed by IHC, such as forSPARC, TOP2A, or PTEN, and used to identify a therapeutic for anindividual. The results of the IHC can be used to form a biomarkerpattern or biomarker signature set. In yet other embodiments, abiological sample from an individual or subject is analyzed for theexpression level of CD52, such as by determining the mRNA expressionlevel by methods including, but not limited to, microarray analysis. Theexpression level of CD52 can be used to identify a therapeutic for theindividual. The expression level of CD52 can be used to form a biomarkerpattern or biomarker signature set. In still other embodiments, thebiomarkers hENT1, cMet, P21, PARP-1, TLE3 and/or IGF1R are assessed toidentify a therapeutic for the individual.

As described herein, the molecular profiling of one or more targets canbe used to determine or identify a therapeutic for an individual. Forexample, the expression level of one or more biomarkers can be used todetermine or identify a therapeutic for an individual. The one or morebiomarkers, such as those disclosed herein, can be used to form abiomarker pattern or biomarker signature set, which is used to identifya therapeutic for an individual. In some embodiments, the therapeuticidentified is one that the individual has not previously been treatedwith. For example, a reference biomarker pattern has been establishedfor a particular therapeutic, such that individuals with the referencebiomarker pattern will be responsive to that therapeutic. An individualwith a biomarker pattern that differs from the reference, for examplethe expression of a gene in the biomarker pattern is changed ordifferent from that of the reference, would not be administered thattherapeutic. In another example, an individual exhibiting a biomarkerpattern that is the same or substantially the same as the reference isadvised to be treated with that therapeutic. In some embodiments, theindividual has not previously been treated with that therapeutic andthus a new therapeutic has been identified for the individual.

Molecular profiling according to the invention can take on abiomarker-centric or a therapeutic-centric point of view. Although theapproaches are not mutually exclusive, the biomarker-centric approachfocuses on sets of biomarkers that are expected to be informative for atumor of a given tumor lineage, whereas the therapeutic-centric pointapproach identifies candidate therapeutics using biomarker panels thatare lineage independent. In a biomarker-centric view, panels of specificbiomarkers are run on different tumor types. This approach provides amethod of identifying a candidate therapeutic by collecting a samplefrom a subject with a cancer of known origin, and performing molecularprofiling on the cancer for specific biomarkers depending on the originof the cancer. The molecular profiling can be performed using any of thevarious techniques disclosed herein. As an example, biomarker panels mayinclude those for breast cancer, ovarian cancer, colorectal cancer, lungcancer, and a “complete” profile to run on any cancer. Markers can beassessed using various techniques such as mutational analysis (e.g.,sequencing approaches), ISH (e.g., FISH/CISH), and for proteinexpression, e.g., using IHC. DNA microarray profiling can be performedon any sample. The candidate therapeutic can be selected based on themolecular profiling results according to the subject methods. Apotential advantage to the bio-marker centric approach is onlyperforming assays that are most likely to yield informative results in agiven lineage. Another postentional advantage is that this approach canfocus on identifying therapeutics conventionally used to treat cancersof the specific lineage. In a therapeutic-centric approach, thebiomarkers assessed are not dependent on the origin of the tumor.Rather, this approach provides a method of identifying a candidatetherapeutic by collecting a sample from a subject with any given cancer,and performing molecular profiling on the cancer for a panel ofbiomarkers without regards to the origin of the cancer. The molecularprofiling can be performed using any of the various techniques disclosedherein, e.g., such as described above. The candidate therapeutic isselected based on the molecular profiling results according to thesubject methods. A potential advantage to the therapeutic-marker centricapproach is that the most promising therapeutics are identified onlytaking into account the molecular characteristics of the tumor itself.Another advantage is that the method can be preferred for a cancer ofunidentified primary origin (CUP). In some embodiments, a hybrid ofbiomarker-centric and therapeutic-centric points of view is used toidentify a candidate therapeutic. This method comprises identifying acandidate therapeutic by collecting a sample from a subject with acancer of known origin, and performing molecular profiling on the cancerfor a comprehensive panel of biomarkers, wherein a portion of themarkers assessed depend on the origin of the cancer. For example,consider a breast cancer. A comprehensive biomarker panel may be run onthe breast cancer, e.g., that for any solid tumor as described herein,but additional sequencing analysis is performed on one or moreadditional markers, e.g., BRCA1 or any other marker with mutationsinformative for theranosis or prognosis of the breast cancer. Theranosiscan be used to refer to the likely efficacy of a therapeutic treatment.Prognosis refers to the likely outcome of an illness. One of skill willapprecitate that the hybrid approach can be used to identify a candidatetherapeutic for any cancer having additional biomarkers that providetheranostic or prognostic information, including the cancers disclosedherein.

Methods for providing a theranosis of disease include selectingcandidate therapeutics for various cancers by assessing a sample from asubject in need thereof (i.e., suffering from a particular cancer). Thesample is assessed by performing an immunohistochemistry (IHC) todetermine of the presence or level of: AR, BCRP, c-KIT, ER, ERCC1, HER2,IGF1R, MET (also referred to herein as cMet), MGMT, MRP1, PDGFR, PGP,PR, PTEN, RRM1, SPARC, TOPO1, TOP2A, TS, COX-2, CK5/6, CK14, CK17, Ki67,p53, CAV-1, CYCLIN D1, EGFR, E-cadherin, p95, TLE3 or a combinationthereof performing a microarray analysis on the sample to determine amicroarray expression profile on one or more (such as at least five, 10,15, 20, 25, 30, 40, 50, 60, 70 or all) of: ABCC1, ABCG2, ADA, AR, ASNS,BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1,DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1,FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP9OAA1, IGFBP3,IGFBP4, IGFBP5, IL2RA, KDR, KIT, LCK, LYN, MET, MGMT, MLH1, MS4A1, MSH2,NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC, PDGFRA, PDGFRB, PGP, PGR,POLA1, PTEN, PTGS2, PTPN12, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG,SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1,TOP2A, TOP2B, TXNRDL TYMS, VDR, VEGFA, VHL, YES1, and ZAP70; comparingthe results obtained from the IHC and microarray analysis against arules database, wherein the rules database comprises a mapping ofcandidate treatments whose biological activity is known against a cancercell that expresses one or more proteins included in the IHC expressionprofile and/or expresses one or more genes included in the microarrayexpression profile; and determining a candidate treatment if thecomparison indicates that the candidate treatment has biologicalactivity against the cancer.

Assessment can further comprise determining a fluorescent in-situhybridization (FISH) profile of EGFR, HER2, cMYC, TOP2A, MET, or acombination thereof, comparing the FISH profile against a rules databasecomprising a mapping of candidate treatments predetermined as effectiveagainst a cancer cell having a mutation profile for EGFR, HER2, cMYC,TOP2A, MET, or a combination thereof, and determining a candidatetreatment if the comparison of the FISH profile against the rulesdatabase indicates that the candidate treatment has biological activityagainst the cancer.

As explained further herein, the FISH analysis can be performed based onthe origin of the sample. This can avoid unnecessary laboratoryprocedures and concomitant expenses by targeting analysis of genes thatare known to play a role in a particular disorder, e.g., a particulartype of cancer. In an embodiment, EGFR, HER2, cMYC, and TOP2A areassessed for breast cancer. In another embodiment, EGFR and MET areassessed for lung cancer. Alternately, FISH analysis of all of EGFR,HER2, cMYC, TOP2A, MET can be performed on a sample. The complete panelmay be assessed, e.g., when a sample is of unknown or mixed origin, toprovide a comprehensive view of an unusual sample, or when economies ofscale dictate that it is more efficient to perform FISH on the entirepanel than to make individual assessments.

In an additional embodiment, the sample is assessed by performingnucleic acid sequencing on the sample to determine a presence of amutation of KRAS, BRAF, NRAS, PIK3CA (also referred to as PI3K), c-Kit,EGFR, or a combination thereof, comparing the results obtained from thesequencing against a rules database comprising a mapping of candidatetreatments predetermined as effective against a cancer cell having amutation profile for KRAS, BRAF, NRAS, PIK3CA, c-Kit, EGFR, or acombination thereof; and determining a candidate treatment if thecomparison of the sequencing to the mutation profile indicates that thecandidate treatment has biological activity against the cancer.

As explained further herein, the nucleic acid sequencing can beperformed based on the origin of the sample. This can avoid unnecessarylaboratory procedures and concomitant expenses by targeting analysis ofgenes that are known to play a role in a particular disorder, e.g., aparticular type of cancer. In an embodiment, the sequences of PIK3CA andc-KIT are assessed for breast cancer. In another embodiment, thesequences of KRAS and BRAF are assessed for GI cancers such ascolorectal cancer. In still another embodiment, the sequences of KRAS,BRAF and EGFR are assessed for lung cancer. Alternately, sequencing ofall of KRAS, BRAF, NRAS, PIK3CA, c-Kit, EGFR can be performed on asample. The complete panel may be sequenced, e.g., when a sample is ofunknown or mixed origin, to provide a comprehensive view of an unusualsample, or when economies of scale dictate that it is more efficient tosequence the entire panel than to make individual assessments.

The genes and gene products used for molecular profiling, e.g., bymicroarray, IHC, FISH, sequencing, and/or PCR (e.g., qPCR), can beselected from those listed in Table 2, Tables 6-9 or Tables 12-15. In anembodiment, IHC is performed for one or more, e.g., 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20 or more, of: AR, BCRP, CAV-1, CD20, CD52, CK 5/6, CK14,CK17, c-kit, CMET, COX-2, Cyclin D1, E-Cad, EGFR, ER, ERCC1, HER-2,IGF1R, Ki67, MGMT, MRP1, P53, p95, PDGFR, PGP, PR, PTEN, RRM1, SPARC,TLE3, TOPO1, TOPO2A, TS, TUBB3; expression analysis (e.g., microarray orRT-PCR) is performed on one or more, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 40, 50 or more, of: ABCC1, ABCG2, ADA, AR, ASNS, BCL2,BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, cKit, c-MYC, DCK, DHFR,DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERCC1, ERCC3, ESR1, FLT1,FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HER2/ERBB2, HIF1A, HSP90,IGFBP3, IGFBP4, IGFBP5, IL2RA, KDR, LCK, LYN, MET, MGMT, MLH1, MS4A1,MSH2, NFKB1, NFKB2, NFKBIA, OGFR, PARP1, PDGFC, PDGFRa, PDGFRA, PDGFRB,PGP, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, ROS1, RRM1, RRM2, RRM2B, RXRB,RXRG, SIK2, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, SPARC, TK1, TNF,TOP2B, TOP2A, TOPO1, TXNRDL TYMS, VDR, VEGFA, VHL, YES1, and ZAP70;fluorescent in-situ hybridization (FISH) is performed on 1, 2, 3, 4, 5,6 or 7 of ALK, cMET, c-MYC, EGFR, HER-2, PIK3CA, and TOPO2A; and DNAsequencing or PCR are performed on 1, 2, 3, 4, 5 or 6 of BRAF, c-kit,EGFR, KRAS, NRAS, and PIK3CA. In an embodiment, all of these genesand/or the gene products thereof are assessed.

Assessing one or more biomarkers disclosed herein can be used forcharacterizing any of the cancers disclosed herein. Characterizingincludes the diagnosis of a disease or condition, the prognosis of adisease or condition, the determination of a disease stage or acondition stage, a drug efficacy, a physiological condition, organdistress or organ rejection, disease or condition progression,therapy-related association to a disease or condition, or a specificphysiological or biological state.

A cancer in a subject can be characterized by obtaining a biologicalsample from a subject and analyzing one or more biomarkers from thesample. For example, characterizing a cancer for a subject or individualmay include detecting a disease or condition (including pre-symptomaticearly stage detecting), determining the prognosis, diagnosis, ortheranosis of a disease or condition, or determining the stage orprogression of a disease or condition. Characterizing a cancer can alsoinclude identifying appropriate treatments or treatment efficacy forspecific diseases, conditions, disease stages and condition stages,predictions and likelihood analysis of disease progression, particularlydisease recurrence, metastatic spread or disease relapse. Characterizingcan also be identifying a distinct type or subtype of a cancer. Theproducts and processes described herein allow assessment of a subject onan individual basis, which can provide benefits of more efficient andeconomical decisions in treatment.

In an aspect, characterizing a cancer includes predicting whether asubject is likely to respond to a treatment for the cancer. As usedherein, a “responder” responds to or is predicted to respond to atreatment and a “non-responder” does not respond or is predicted to notrespond to the treatment. Biomarkers can be analyzed in the subject andcompared to biomarker profiles of previous subjects that were known torespond or not to a treatment. If the biomarker profile in a subjectmore closely aligns with that of previous subjects that were known torespond to the treatment, the subject can be characterized, orpredicted, as a responder to the treatment. Similarly, if the biomarkerprofile in the subject more closely aligns with that of previoussubjects that did not respond to the treatment, the subject can becharacterized, or predicted as a non-responder to the treatment.

The sample used for characterizing a cancer can be any disclosed herein,including without limitation a tissue sample, tumor sample, or a bodilyfluid. Bodily fluids that can be used included without limitationperipheral blood, sera, plasma, ascites, urine, cerebrospinal fluid(CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor,amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid,semen (including prostatic fluid), Cowper's fluid or pre-ejaculatoryfluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid,pleural and peritoneal fluid, pericardial fluid, malignant effusion,lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum,vomit, vaginal secretions, mucosal secretion, stool water, pancreaticjuice, lavage fluids from sinus cavities, bronchopulmonary aspirates orother lavage fluids. In an embodiment, the sample comprises vesicles.The biomarkers can be associated with the vesicles. In some embodiments,vesicles are isolated from the sample and the biomarkers associated withthe vesicles are assessed.

Comprehensive and Standard-of-Care Molecular Profiling

Molecular profiling according to the invention can be used to guidetreatment selection for cancers at any stage of disease or priortreatment. Molecular profiling comprises assessment of DNA mutations,gene rearrangements, gene copy number variation, RNA expression, proteinexpression, as well as assessment of other biological entities andphenomena that can inform clinical decision making. In some embodiments,the methods herein are used to guide selection of candidate treatmentsusing the standard of care treatments for a particular type or lineageof cancer. Profiling of biomarkers that implicate standard-of-caretreatments may be used to assist in treatment selection for a newlydiagnosed cancer having multiple treatment options. Such profiling maybe referred to herein as “select” profiling. Standard-of-care treatmentsmay comprise NCCN on-compendium treatments or other standard treatmentsused for a cancer of a given lineage. One of skill will appreciate thatsuch profiles can be updated as the standard of care and/or availabilityof experimental agents for a given disease lineage change. In otherembodiments, molecular profiling is performed for additional biomarkersto identify treatments as beneficial or not beyond that go beyond thestandard-of-care for a particular lineage or stage of the cancer. Such“comprehensive” profiling can be performed to assess a wide panel ofdruggable or drug-associated biomarker targets for any biological sampleor specimen of interest. One of skill will appreciate that the selectprofiles generally comprise subsets of the comprehensive profile. Thecomprehensive profile can also be used to guide selection of candidatetreatments for any cancer at any point of care. The comprehensiveprofile may also be preferable when standard-of-care treatments notexpected to provide further benefit, such as in the salvage treatmentsetting for recurrent cancer or wherein all standard treatments havebeen exhausted. For example, the comprehensive profile may be used toassist in treatment selection when standard therapies are not an optionfor any reason including, without limitation, when standard treatmentshave been exhausted for the patient. The comprehensive profile may beused to assist in treatment selection for highly aggressive or raretumors with uncertain treatment regimens. For example, a comprehensiveprofile can be used to identify a candidate treatment for a newlydiagnosed case or when the patient has exhausted standard of caretherapies or has an aggressive disease. In practice, molecular profilingaccording to the invention has indeed identified beneficial therapiesfor a cancer patient when all standard-of-care treatments were exhaustedthe treating physician was unsure ofwhat treatment to select next. Seethe Examples herein. One of skill in the art will appreciate that by itsvery nature a comprehensive molecular profiling can be used to select atherapy for any appropriate indication independent of the nature of theindication (e.g., source, stage, prior treatment, etc). However, in someembodiments, a comprehensive molecular profile is tailored for aparticular indication. For example, biomarkers associated withtreatments that are known to be ineffective for a cancer from aparticular lineage or anatomical origin may not be assessed as part of acomprehensive molecular profile for that particular cancer. Similarly,biomarkers associated with treatments that have been previously used andfailed for a particular patient may not be assessed as part of acomprehensive molecular profile for that particular patient. In yetanother non-limiting example, biomarkers associated with treatments thatare only known to be effective for a cancer from a particular anatomicalorigin may only be assessed as part of a comprehensive molecular profilefor that particular cancer. One of skill will further appreciate thatthe comprehensive molecular profile can be updated to reflectadvancements, e.g., new treatments, new biomarker-drug associations, andthe like, as available.

Molecular Intelligence Profiles

The invention provides molecular intelligence (MI) molecular profilesusing a variety of techniques to assess panels of biomarkers in order toselect or not select a candidate therapeutic for treating a cancer. Suchtechniques comprise IHC for expression profiling, CISH/FISH for DNA copynumber, and Sanger, Pyrosequencing, PCR, RFLP, fragment analysis andNext Generation sequencing for mutational analysis. Exemplary profilesare described in Tables 7-8 herein. The profiling can be performed usingthe biomarker-drug associations and related rules for the various cancerlineages as described, e.g., in any one of Tables 3-6, Tables 9-10,Table 17, and Tables 22-24. In some embodiments, the associations areaccording to Tables 6 and/or 9. Additional biomarker—drug associationscan be found in the following International Patent Applications, each ofwhich is incorporated herein by reference in its entirety:PCT/US2007/69286, filed May 18, 2007; PCT/US2009/60630, filed Oct. 14,2009; PCT/2010/000407, filed Feb. 11, 2010; PCT/US12/41393, filed Jun.7, 2012; PCT/US2013/073184, filed Dec. 4, 2013; PCT/US2010/54366, filedOct. 27, 2010; PCT/US11/67527, filed Dec. 28, 2011; and PCT/US15/13618,filed Jan. 29, 2015. Molecular intelligence profiles may includeanalysis of a panel of genes linked to known therapies and clinicaltrials, as well as genes that are known to be involved in cancer andhave alternative clinical utilities including predictive, prognostic ordiagnostic uses, as shown in Table 8. The panel may be assessed usingNext Generation sequencing analysis. In some cases, the MI molecularprofiles include analysis of an expanded panel of genes such as inTables 12-15.

The biomarkers which comprise the molecular intelligence molecularprofiles can include genes or gene products that are known to beassociated directly with a particular drug or class of drugs. Thebiomarkers can also be genes or gene products that interact with suchdrug associated targets, e.g., as members of a common pathway. Thebiomarkers can be selected from Table 2. In some embodiments, the genesand/or gene products included in the molecular intelligence (MI)molecular profiles are selected from Table 6. For example, the molecularprofiles can be performed for at least one, e.g., at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or 76 of1p19q, ABL1, AKT1, ALK, APC, AR, AREG, ATM, BRAF, BRCA1, BRCA2, CDH1,CSF1R, CTNNB1, EGFR, EGFRvIII, ER, ERBB2, ERBB3, ERBB4, ERCC1, EREG,FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, H3K36me3, HNF1A, HRAS,IDH1, IDH2, JAK2, JAK3, KDR, KIT (cKit), KRAS, MET (cMET), MGMT, MLH1,MPL, MSH2, MSH6, MSI, NOTCH1, NPM1, NRAS, PBRM1, PDGFRA, PD-1, PD-L1,PGP, PIK3CA (PI3K), PMS2, PR, PTEN, PTPN11, RB1, RET, ROS1, RRM1, SMAD4,SMARCB1, SMO, SPARC, STK11, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL,and VEGFR2. The biomarkers can be assessed using the laboratory methodsas listed in Tables 7-8, or using similar analysis methodology such asdisclosed herein.

TABLE 6 Exemplary Genes and Gene Products and Related Therapies 1p19q1p19q codeletions result from an unbalanced translocation between the pand q arms in chromosomes 1 and 19, respectively. Along with IDHmutations, 1p19q deletions are associated with oligodendrogliomatumorigenesis. Rates of 1p19q codeletion are especially high inlow-grade and anaplastic oligodendroglioma. By contrast, 1p19qcodeletions are lower in high grade gliomas like anaplastic astrocytomaand glioblastoma multiforme. NCCN Central Nervous System Guidelinesmention 1p19q codeletions are indicative of a better prognosis inoligodendroglioma. Prospective studies indicate 1p19q codeletions areassociated with potential benefit to PCV (procarbazine, CCNU[lomustine], vincristine) chemotherapy in anaplastic oligodendroglialtumors. ABL1 ABL1 also known as Abelson murine leukemia homolog 1. MostCML patients have a chromosomal abnormality due to a fusion betweenAbelson (Abl) tyrosine kinase gene at chromosome 9 and break pointcluster (Bcr) gene at chromosome 22 resulting in constitutive activationof the Bcr-Abl fusion gene. Imatinib is a Bcr-Abl tyrosine kinaseinhibitor commonly used in treating CML patients. Mutations in the ABL1gene are common in imatinib resistant CML patients which occur in 30-90%of patients. However, more than 50 different point mutations in the ABL1kinase domain may be inhibited by the second generation kinaseinhibitors, dasatinib, bosutinib and nilotinib. The gatekeeper mutation,T315I that causes resistance to all currently approved TKIs accounts forabout 15% of the mutations found in patients with imatinib resistance.BCR-ABL1 mutation analysis is recommended to help facilitate selectionof appropriate therapy for patients with CML after treatment withimatinib fails. AKT1 AKT1 gene (v-akt murine thymoma viral oncogenehomologue 1) encodes a serine/threonine kinase which is a pivotalmediator of the PI3K-related signaling pathway, affecting cell survival,proliferation and invasion. Dysregulated AKT activity is a frequentgenetic defect implicated in tumorigenesis and has been indicated to bedetrimental to hematopoiesis. Activating mutation E17K has beendescribed in breast (2-4%), endometrial (2-4%), bladder cancers (3%),NSCLC (1%), squamous cell carcinoma of the lung (5%) and ovarian cancer(2%). This mutation in the pleckstrin homology domain facilitates therecruitment of AKT to the plasma membrane and subsequent activation byaltering phosphoinositide binding. A mosaic activating mutation E17K hasalso been suggested to be the cause of Proteus syndrome. Mutation E49Khas been found in bladder cancer, which enhances AKT activation andshows transforming activity in cell lines. ALK ALK or anaplasticlymphoma receptor tyrosine kinase belongs to the insulin receptorsuperfamily. It has been found to be rearranged or mutated in tumorsincluding anaplastic large cell lymphomas, neuroblastoma, anaplasticthyroid cancer and non-small cell lung cancer. EML4-ALK fusion or pointmutations of ALK result in the constitutively active ALK kinase, causingaberrant activation of downstream signaling pathways including RAS-ERK,JAK3-STAT3 and PI3K-AKT. Patients with an EML4-ALK rearrangement arelikely to respond to the ALK-targeted agent crizotinib and ceritinib.ALK secondary mutations found in NSCLC have been associated withacquired resistance to ALK inhibitor, crizotinib and ceritinib. AR Theandrogen receptor (AR) gene encodes for the androgen receptor protein, amember of the steroid receptor family. Like other members of the nuclearsteroid receptor family, AR is a DNA-binding transcription factoractivated by specific hormones, in this case testosterone or DHT.Mutations of this gene are not often found in untreated, localizedprostate cancer. Instead, they occur more frequently inhormone-refractory, androgen- ablated, and metastatic tumors. Recentfindings indicate that specific mutations in AR (e.g. F876L, AR-V7) areassociated with resistance to newer-generation, AR-targeted therapiessuch as enzalutamide. APC APC or adenomatous polyposis coli is a keytumor suppressor gene that encodes for a large multi-domain protein.This protein exerts its tumor suppressor function in the Wnt/ß- catenincascade mainly by controlling the degradation of ß-catenin, the centralactivator of transcription in the Wnt signaling pathway. The Wntsignaling pathway mediates important cellular functions includingintercellular adhesion, stabilization of the cytoskeleton, and cellcycle regulation and apoptosis, and it is important in embryonicdevelopment and oncogenesis. Mutation in APC results in a truncatedprotein product with abnormal function, lacking the domains involved inß-catenin degradation. Somatic mutation in the APC gene can be detectedin the majority of colorectal tumors (80%) and it is an early event incolorectal tumorigenesis. APC wild type patients have shown betterdisease control rate in the metastatic setting when treated withoxaliplatin, while when treated with fluoropyrimidine regimens, APC wildtype patients experience more hematological toxicities. APC mutation hasalso been identified in oral squamous cell carcinoma, gastric cancer aswell as hepatoblastoma and may contribute to cancer formation. Germlinemutation in APC causes familial adenomatous polyposis, which is anautosomal dominant inherited disease that will inevitably develop tocolorectal cancer if left untreated. COX-2 inhibitors includingcelecoxib may reduce the recurrence of adenomas and incidence ofadvanced adenomas in individuals with an increased risk of CRC. Turcotsyndrome and Gardner's syndrome have also been associated with germlineAPC defects. Germline mutations of the APC have also been associatedwith an increased risk of developing desmoid disease, papillary thyroidcarcinoma and hepatoblastoma. AREG AREG, also known as amphiregulin, isa ligand of the epidermal growth factor receptor. Overexpression of AREGin primary colorectal cancer patients has been associated with increasedclinical benefit from cetuximab in KRAS wildtype patients. ATM ATM orataxia telangiectasia mutated is activated by DNA double-strand breaksand DNA replication stress. It encodes a protein kinase that acts as atumor suppressor and regulates various biomarkers involved in DNArepair, which include p53, BRCA1, CHK2, RAD17, RAD9, and NBS1. AlthoughATM is associated with hematologic malignancies, somatic mutations havebeen found in colon (18%), head and neck (14%), and prostate (12%)cancers. Inactivating ATM mutations make patients potentially moresusceptible to PARP inhibitors. Germline mutations in ATM are associatedwith ataxia-telangiectasia (also known as Louis-Bar syndrome) and apredisposition to malignancy. BRAF BRAF encodes a protein belonging tothe raf/mil family of serine/threonine protein kinases. This proteinplays a role in regulating the MAP kinase/ERK signaling pathwayinitiated by EGFR activation, which affects cell division,differentiation, and secretion. BRAF somatic mutations have been foundin melanoma (43%), thyroid (39%), biliary tree (14%), colon (12%), andovarian tumors (12%). A BRAF enzyme inhibitor, vemurafenib, was approvedby FDA to treat unresectable or metastatic melanoma patients harboringBRAF V600E mutations. BRAF inherited mutations are associated withNoonan/Cardio- Facio-Cutaneous (CFC) syndrome, syndromes associated withshort stature, distinct facial features, and potential heart/skeletalabnormalities. BRCA1 BRCA1 or breast cancer type 1 susceptibility geneencodes a protein involved in cell growth, cell division, and DNA-damagerepair. It is a tumor suppressor gene which plays an important role inmediating double-strand DNA breaks by homologous recombination (HR).Tumors with BRCA1 mutation may be more sensitive to platinum agents andPARP inhibitors. BRCA2 BRCA2 or breast cancer type 2 susceptibility geneencodes a protein involved in cell growth, cell division, and DNA-damagerepair. It is a tumor suppressor gene which plays an important role inmediating double-strand DNA breaks by homologous recombination (HR).Tumors with BRCA2 mutation may be more sensitive to platinum agents andPARP inhibitors. CDH1 This gene is a classical cadherin from thecadherin superfamily. The encoded protein is a calcium dependentcell-cell adhesion glycoprotein comprised of five extracellular cadherinrepeats, a transmembrane region and a highly conserved cytoplasmic tail.The protein plays a major role in epithelial architecture, cell adhesionand cell invasion. Mutations in this gene are correlated with gastric,breast, colorectal, thyroid and ovarian cancer. Loss of function isthought to contribute to progression in cancer by increasingproliferation, invasion, and/or metastasis. The ectodomain of thisprotein mediates bacterial adhesion to mammalian cells and thecytoplasmic domain is required for internalization. CSF1R CSF1R orcolony stimulating factor 1 receptor gene encodes a transmembranetyrosine kinase, a member of the CSF1/PDGF receptor family. CSF1Rmediates the cytokine (CSF- 1) responsible for macrophage production,differentiation, and function. Although associated with hematologicmalignancies, mutations of this gene are associated with cancers of theliver (21%), colon (13%), prostate (3%), endometrium (2%), and ovary(2%). It is suggested that patients with CSF1R mutations could respondto imatinib. Germline mutations in CSF1R are associated with diffuseleukoencephalopathy, a rapidly progressive neurodegenerative disorder.CTNNB1 CTNNB1 or cadherin-associated protein, beta 1, encodes forß-catenin, a central mediator of the Wnt signaling pathway whichregulates cell growth, migration, differentiation and apoptosis.Mutations in CTNNB1 (often occurring in exon 3) prevent the breakdown ofß- catenin, which allows the protein to accumulate resulting inpersistent transactivation of target genes, including c-myc andcyclin-D1. Somatic CTNNB1 mutations occur in 1-4% of colorectal cancers,2-3% of melanomas, 25-38% of endometrioid ovarian cancers, 84- 87% ofsporadic desmoid tumors, as well as the pediatric cancers,hepatoblastoma, medulloblastoma and Wilms' tumors. EGFR EGFR orepidermal growth factor receptor, is a transmembrane receptor tyrosinekinase belonging to the ErbB family of receptors. Upon ligand binding,the activated receptor triggers a series of intracellular pathways(Ras/MAPK, PI3K/Akt, JAK-STAT) that result in cell proliferation,migration and adhesion. EGFR mutations have been observed in 20- 25% ofnon-small cell lung cancer (NSCLC), 10% of endometrial and peritonealcancers. Somatic gain-of-function EGFR mutations, including in-framedeletions in exon 19 or point mutations in exon 21, confer sensitivityto first- and second-generation tyrosine kinase inhibitors (TKIs, e.g.,erlotinib, gefitinib and afatinib), whereas the secondary mutation,T790M in exon 20, confers reduced response. Non-small cell lung cancercancer patients overexpressing EGFR protein have been found to respondto the EGFR monoclonal antibody, cetuximab. Germline mutations andpolymorphisms of EGFR have been associated with familial lungadenocarcinomas. EGFRvIII EGFRvIII is a mutated form of EGFR withdeletion of exon 2 to 7 on the extracellular ligand-binding domain. Thisgenetic alteration has been found in about 30% of glioblastoma, 30% ofhead and neck squamous cell cancer, 30% of breast cancer and 15% ofNSCLC, and has not been found in normal tissue. EGFRvIII can formhomo-dimers or heterodimers with EGFR or ERBB2, resulting inconstitutive activation in the absence of ligand binding, activatingvarious downstream signaling pathways including the PI3K and MAPKpathways, leading to increased cell proliferation and motility as wellas inhibition of apoptosis. Preliminary studies have shown that EGFRvIIIexpression may associate with higher sensitivity to erlotinib andgefitinib, as well as to pan-Her inhibitors including neratinib anddacomitinib. EGFRvIII peptide vaccine rindopepimut (CDX-110) andmonoclonal antibodies specific to EGFRvIII including ABT-806 and AMG595are being investigated in clinical trials. ER The estrogen receptor (ER)is a member of the nuclear hormone family of intracellular receptorswhich is activated by the hormone estrogen. It functions as a DNAbinding transcription factor to regulate estrogen-mediated geneexpression. Estrogen receptors overexpressing breast cancers arereferred to as ‘ER positive.’ Estrogen binding to ER on cancer cellsleads to cancer cell proliferation. Breast tumors over-expressing ER aretreated with hormone-based anti-estrogen therapy. For example,everolimus combined with exemestane may improve survival in ER positiveHer2 negative breast cancer patients who are resistant to aromataseinhibitors. ERBB2 ERBB2 (HER2 (human epidermal growth factor receptor2)) or v-erb-b2 erythroblastic leukemia viral oncogene homolog 2,encodes a member of the epidermal growth factor (EGF) receptor family ofreceptor tyrosine kinases. This gene binds to other ligand-bound EGFreceptor family members to form a heterodimer and enhanceskinase-mediated activation of downstream signaling pathways, leading tocell proliferation. Most common mechanism for activation of HER2 aregene amplification and over-expression with somatic mutations beingrare. Her2 is overexpressed in 15-30% of newly diagnosed breast cancers.Clinically, Her2 is a target for the monoclonal antibodies trastuzumaband pertuzumab which bind to the receptor extracellularly; the kinaseinhibitor lapatinib binds and blocks the receptor intracellularly. ERBB3ERBB3 encodes a protein (HER3 (human epidermal growth factor receptor3)) that is a member of the EGFR family of protein tyrosine kinases.ERBB3 protein does not actually contain a kinase domain itself, but itcan activate other members of the EGFR kinase family by formingheterodimers. Heterodimerization with other kinases triggers anintracellular cascade increasing cell proliferation. Mutations in ERBB3have been observed primarily in gastric cancer and cancer of the gallbladder. Other tissue types known to harbor ERBB3 mutations includehormone-positive breast cancer, glioblastoma, ovarian, colon, head andneck and lung. ERBB4 ERBB4 (HER4) is a member of the Erbb receptorfamily known to play a pivotal role in cell-cell signaling and signaltransduction regulating cell growth and development. The most commonlyaffected signaling pathways are the PI3K-Akt and MAP kinase pathways.Erbb4 was found to be somatically mutated in 19% of melanomas and Erbb4mutations may confer “oncogene addiction” on melanoma cells. Erbb4mutations have also been observed in various other cancer types,including, gastric carcinomas (2%), colorectal carcinomas (1-3%),non-small cell lung cancer (2-5%) and breast carcinomas (1%). ERCC1ERCC1, or excision repair cross-complementation group 1, is a keycomponent of the nucleotide excision repair (NER) pathway. NER is a DNArepair mechanism necessary for the repair of DNA damage from a varietyof sources including platinum agents. Tumors with low expression ofERCC1 have impaired NER capacity and may be more sensitive to platinumagents. EREG EREG, also known as epiregulin, is a ligand of theepidermal growth factor receptor. Overexpression of EREG in primarycolorectal cancer patients has been related to clinical outcome in KRASwildtype patients treated with cetuximab indicating ligand drivenautocrine oncogenic EGFR signaling. FBXW7 FBXW7 or E3 ligase F-box andWD repeat domain containing 7, also known as Cdc4, encodes three proteinisoforms which constitute a component of the ubiquitin-proteasomecomplex. Mutation of FBXW7 occurs in hotspots and disrupts therecognition of and binding with substrates which inhibits the propertargeting of proteins for degradation (e.g. Cyclin E, c-Myc, SREBP1,c-Jun, Notch-1, mTOR and MCL1). Mutation frequencies identified incholangiocarcinomas, acute T-lymphoblastic leukemia/lymphoma, andcarcinomas of endometrium, colon and stomach are 35%, 31%, 9%, 9%, and6%, respectively. Targeting an oncoprotein downstream of FBXW7, such asmTOR or c-Myc, may provide a therapeutic strategy. Tumor cells withmutated FBXW7 may be sensitive to rapamycin treatment, suggesting FBXW7loss (mutation) may be a predictive biomarker for treatment withinhibitors of the mTOR pathway. In addition, it has been proposed thatloss of FBXW7 confers resistance to tubulin-targeting agents likepaclitaxel or vinorelbine, by interfering with the degradation of MCL1,a regulator of apoptosis. FGFR1 FGFR1 or fibroblast growth factorreceptor 1, encodes for FGFR1 which is important for cell division,regulation of cell maturation, formation of blood vessels, wound healingand embryonic development. Somatic activating mutations are rare, buthave been documented in melanoma, glioblastoma, and lung tumors.Germline, gain-of-function mutations in FGFR1 result in developmentaldisorders including Kallmann syndrome and Pfeiffer syndrome. Preclinicalstudies suggest that FGFR1 amplification may be associated withendocrine resistance in breast cancer. FGFR1 amplification has beenobserved in various cancer types including breast cancer, squamous celllung cancer, head and neck squamous cell cancer and esophageal cancerand may indicate sensitivity to FGFR-targeted therapies. FGFR2 FGFR2 isa receptor for fibroblast growth factor. Activation of FGFR2 throughmutation and amplification has been noted in a number of cancers.Somatic mutations of the fibroblast growth factor receptor 2 (FGFR2)tyrosine kinase are present in endometrial carcinoma, lung squamous cellcarcinoma, cervical carcinoma, and melanoma. In the endometrioidhistology of endometrial cancer, the frequency of FGFR2 mutation is 16%and the mutation is associated with shorter disease free survival inpatients diagnosed with early stage disease. Loss of function FGFR2mutations occur in about 8% melanomas and contribute to melanomapathogenesis. Germline mutations in FGFR2 are associated with numerousmedical conditions that include congenital craniofacial malformationdisorders, Apert syndrome and the related Pfeiffer and Crouzonsyndromes. Amplification of FGFR2 has been shown in 5-10% of gastriccancer and breast cancer and may indicate sensitivity to FGFR-targetedtherapies. FLT3 FLT3 or Fms-like tyrosine kinase 3 receptor is a memberof class III receptor tyrosine kinase family, which includes PDGFRA/Band KIT. Signaling through FLT3 ligand- receptor complex regulateshematopoiesis, specifically lymphocyte development. The FLT3 internaltandem duplication (FLT3-ITD) is the most common genetic lesion in acutemyeloid leukemia (AML), occurring in 25% of cases. FLT3 mutations areuncommon in solid tumors; however they have been documented in breastcancer. GNA11 GNA11 is a proto-oncogene that belongs to the Gq family ofthe G alpha family of G protein coupled receptors. Known downstreamsignaling partners of GNA11 are phospholipase C beta and RhoA andactivation of GNA11 induces MAPK activity. Over half of uveal melanomapatients lacking a mutation in GNAQ exhibit somatic mutations in GNA11.Activating mutations of GNA11 have not been found in other malignancies.GNAQ This gene encodes the Gq alpha subunit of G proteins. G proteinsare a family of heterotrimeric proteins coupling seven-transmembranedomain receptors. Oncogenic mutations in GNAQ result in a loss ofintrinsic GTPase activity, resulting in a constitutively active Galphasubunit. This results in increased signaling through the MAPK pathway.Somatic mutations in GNAQ have been found in 50% of primary uvealmelanoma patients and up to 28% of uveal melanoma metastases. GNAS GNAS(or GNAS complex locus) encodes a stimulatory G protein alpha-subunit.These guanine nucleotide binding proteins (G proteins) are a family ofheterotrimeric proteins which couple seven-transmembrane domainreceptors to intracellular cascades. Stimulatory G-protein alpha-subunittransmits hormonal and growth factor signals to effector proteins and isinvolved in the activation of adenylate cyclases. Mutations of GNAS geneat codons 201 or 227 lead to constitutive cAMP signaling. GNAS somaticmutations have been found in pituitary (28%), pancreatic (20%), ovarian(11%), adrenal gland (6%), and colon (6%) cancers. Patients with somaticGNAS mutations may derive benefit from clinical trials with MEKinhibitors. Germline mutations of GNAS have been shown to be the causeof McCune-Albright syndrome (MAS), a disorder marked by endocrine,dermatologic, and bone abnormalities. GNAS is usually found as a mosaicmutation in patients. Loss of function mutations are associated withpseudohypoparathyroidism and pseudopseudohypoparathyroidism. H3K36me3Trimethylated histone H3 lysine 36 (H3K36me3) is a chromatin regulatoryprotein that regulates gene expression. A loss of H3K36me3 proteincorrelates with loss of expression or mutation of SETD2 which is amember of the SET domain family of histone methyltransferases. Loss ofSETD2 as well as H3K36m3 protein has been detected in various solidtumors including renal cell carcinoma and breast cancer and leads topoor prognosis. HRAS HRAS (homologous to the oncogene of the Harvey ratsarcoma virus), together with KRAS and NRAS, belong to the superfamilyof RAS GTPase. RAS protein activates RAS- MEK-ERK/MAPK kinase cascadeand controls intracellular signaling pathways involved in fundamentalcellular processes such as proliferation, differentiation, andapoptosis. Mutant Ras proteins are persistently GTP-bound and active,causing severe dysregulation of the effector signaling HRAS mutationshave been identified in cancers from the urinary tract (10%-40%), skin(6%) and thyroid (4%) and they account for 3% of all RAS mutationsidentified in cancer. RAS mutations (especially HRAS mutations) occur(5%) in cutaneous squamous cell carcinomas and keratoacanthomas thatdevelop in patients treated with BRAF inhibitor vemurafenib, likely dueto the paradoxical activation of the MAPK pathway. Germline mutation inHRAS has been associated with Costello syndrome, a genetic disorder thatis characterized by delayed development and mental retardation anddistinctive facial features and heart abnormalities. IDH1 IDH1 encodesfor isocitrate dehydrogenase in cytoplasm and is found to be mutated in60- 90% of secondary gliomas, 75% of cartilaginous tumors, 17% ofthyroid tumors, 15% of cholangiocarcinoma, 12-18% of patients with acutemyeloid leukemia, 5% of primary gliomas, 3% of prostate cancer, as wellas in less than 2% in paragangliomas, colorectal cancer and melanoma.Mutated IDH1 results in impaired catalytic function of the enzyme, thusaltering normal physiology of cellular respiration and metabolism. IDH2IDH2 encodes for the mitochondrial form of isocitrate dehydrogenase, akey enzyme in the citric acid cycle, which is essential for cellrespiration. Mutation in IDH2 not only results in impaired catalyticfunction of the enzyme, but also causes the overproduction of anonco-metabolite, 2-hydroxy-glutarate, which can extensively alter themethylation profile in cancer. IDH2 mutation is mutually exclusive ofIDH1 mutation, and has been found in 2% of gliomas and 10% of AML, aswell as in cartilaginous tumors and cholangiocarcinoma. In gliomas, IDH2mutations are associated with lower grade astrocytomas,oligodendrogliomas (grade II/III), as well as secondary glioblastoma(transformed from a lower grade glioma), and are associated with abetter prognosis. In secondary glioblastoma, preliminary evidencesuggests that IDH2 mutation may associate with a better response toalkylating agent temozolomide. IDH mutations have also been suggested toassociate with a benefit from using hypomethylating agents in cancersincluding AML. Germline IDH2 mutation has been indicated to associatewith a rare inherited neurometabolic disorder D-2-hydroxyglutaricaciduria. JAK2 JAK2 or Janus kinase 2 is a part of the JAK/STAT pathwaywhich mediates multiple cellular responses to cytokines and growthfactors including proliferation and cell survival. It is also essentialfor numerous developmental and homeostatic processes, includinghematopoiesis and immune cell development. Mutations in the JAK2 kinasedomain result in constitutive activation of the kinase and thedevelopment of chronic myeloproliferative neoplasms such as polycythemiavera (95%), essential thrombocythemia (50%) and myelofibrosis (50%).JAK2 mutations were also found in BCR-ABL1-negative acute lymphoblasticleukemia patients and the mutated patients show a poor outcome. Germlinemutations in JAK2 have been associated with myeloproliferative neoplasmsand thrombocythemia. JAK3 JAK3 or Janus activated kinase 3 is anintracellular tyrosine kinase involved in cytokine signaling, whileinteracting with members of the STAT family. Like JAK1, JAK2, and TYK2,JAK3 is a member of the JAK family of kinases. When activated, kinaseenzymes phosphorylate one or more signal transducer and activator oftranscription (STAT) factors, which translocate to the cell nucleus andregulate the expression of genes associated with survival andproliferation. JAK3 signaling is related to T cell development andproliferation. This biomarker is found in malignancies including withoutlimitation head and neck (21%) colon (7%), prostate (5%), ovary (4%),breast (2%), lung (1%), and stomach (1%) cancer. Its prognostic andpredictive utility is under investigation. Germline mutations of JAK3are associated with severe, combined immunodeficiency disease (SCID).KDR KDR (kinase insert domain receptor), also known as VEGFR2 (vascularendothelial growth factor 2), is one of three main subtypes of VEGFR andis expressed on almost all endothelial cells. This protein is animportant signaling protein in angiogenesis. VEGFR2 copy number changesare frequently observed in lung, glioma and triple negative breastcancer. Evidence suggests that increased levels of VEGFR2 may bepredictive of response to anti-angiogenic drugs and multi-targetedkinase inhibitors. Several VEGFR antagonists are either FDA-approved orin clinical trials (i.e. bevacizumab, cabozantinib, regorafenib,pazopanib, and vandetanib). KIT (cKit) c-KIT is a receptor tyrosinekinase expressed by hematopoietic stem cells, interstitial cells ofcajal (pacemaker cells of the gut) and other cell types. Upon binding ofc-KIT to stem cell factor (SCF), receptor dimerization initiates aphosphorylation cascade resulting in proliferation, apoptosis,chemotaxis and adhesion. C-KIT mutation has been identified in variouscancer types including gastrointestinal stromal tumors (GIST) (up to85%) and melanoma (chronic sun damage type, acral or mucosal) (20-40%).C-KIT is inhibited by multi-targeted agents including imatinib andsunitinib. KRAS KRAS or V-Ki-ras2 Kirsten rat sarcoma viral oncogenehomolog encodes a signaling intermediate involved in many signalingcascades including the EGFR pathway. KRAS somatic mutations have beenfound in pancreatic (57%), colon (35%), lung (16%), biliary tract (28%),and endometrial (15%) cancers. Mutations at activating hotspots areassociated with resistance to EGFR tyrosine kinase inhibitors(erlotinib, gefitinib) in NSCLC and monoclonal antibodies (cetuximab,panitumumab) in CRC patients. Patients with KRAS G13D mutation have beenshown to derive benefit from anti-EGFR monoclonal antibody therapy inCRC patients. Several germline mutations of KRAS (V14I, T58I, and D153Vamino acid substitutions) are associated with Noonan syndrome. MET(cMET) MET is a proto-oncogene that encodes the tyrosine kinasereceptor, cMET, of hepatocyte growth factor (HGF) or scatter factor(SF). cMet mutations cause aberrant MET signaling in various cancertypes including renal papillary, hepatocellular, head and neck squamous,gastric carcinomas and non-small cell lung cancer. Specifically,mutations in the juxtamembrane domain (exon 14, 15) results in theconstitutive activation and show enhanced tumorigenicity. Germlinemutations in cMET have been associated with hereditary papillary renalcell carcinoma. MGMT O-6-methylguanine-DNA methyltransferase (MGMT)encodes a DNA repair enzyme. MGMT expression is mainly regulated at theepigenetic level through CpG island promoter methylation which in turncauses functional silencing of the gene. MGMT methylation and/or lowexpression has been correlated with response to alkylating agents liketemozolomide and dacarbazine. MLH1 MLH1 or mutL homolog 1, colon cancer,nonpolyposis type 2 (E. coli) gene encodes a mismatch repair (MMR)protein which repairs DNA mismatches that occur during replication.Although the frequency is higher in colon cancer (10%), MLH1 somaticmutations have been found in esophageal (6%), ovarian (5%), urinarytract (5%), pancreatic (5%), and prostate (5%) cancers. Germlinemutations of MLH1 are associated with Lynch syndrome, also known ashereditary non-polyposis colorectal cancer (HNPCC). Patients with Lynchsyndrome are at increased risk for various malignancies, includingintestinal, gynecologic, and upper urinary tract cancers and in itsvariant, Muir- Torre syndrome, with sebaceous tumors. MPL MPL ormyeloproliferative leukemia gene encodes the thrombopoietin receptor,which is the main humoral regulator of thrombopoiesis in humans. MPLmutations cause constitutive activation of JAK-STAT signaling and havebeen detected in 5-7% of patients with primary myelofibrosis (PMF) and1% of those with essential thrombocythemia (ET). MSH2 This locus isfrequently mutated in hereditary nonpolyposis colon cancer (HNPCC). Whencloned, it was discovered to be a human homolog of the E. coli mismatchrepair gene mutS, consistent with the characteristic alterations inmicrosatellite sequences found in HNPCC. The protein product is acomponent of the DNA mismatch repair system (MMR), and forms twodifferent heterodimers: MutS alpha (MSH2-MSH6 heterodimer) and MutS beta(MSH2-MSH3 heterodimer) which binds to DNA mismatches thereby initiatingDNA repair. After mismatch binding, MutS alpha or beta forms a ternarycomplex with the MutL alpha heterodimer, which is thought to beresponsible for directing the downstream MMR events. MutS alpha may alsoplay a role in DNA homologous recombination repair. MSH6 This geneencodes a member of the DNA mismatch repair MutS family. Mutations inthis gene may be associated with hereditary nonpolyposis colon cancer,colorectal cancer, and endometrial cancer. The protein product is acomponent of the DNA mismatch repair system (MMR), and heterodimerizeswith MSH2 to form MutS alpha, which binds to DNA mismatches therebyinitiating DNA repair. MutS alpha may also play a role in DNA homologousrecombination repair. Recruited on chromatin in G1 and early S phase viaits PWWP domain that specifically binds trimethylated ‘Lys-36’ ofhistone H3 (H3K36me3): early recruitment to chromatin to be replicatedallowing a quick identification of mismatch repair to initiate the DNAmismatch repair reaction. MSI Microsatellites are short, tandem repeatedDNA sequences from 1-6 base pairs in length. These repeats aredistributed throughout the genome and often vary in length from oneindividual to another due to differences in the number of tandem repeatsat each locus. They can be used to detect a form of genomic instabilitycalled microsatellite instability. MSI is a change in length of amicrosatellite allele due to insertion or deletion of repeat unitsduring DNA replication and failure of the DNA mis-match repair system tocorrect these errors. Therefore, the presence of MSI is indicative of aloss of mismatch repair (MMR) activity. NOTCH1 NOTCH1 or notch homolog1, translocation-associated, encodes a member of the Notch signalingnetwork, an evolutionary conserved pathway that regulates developmentalprocesses by regulating interactions between physically adjacent cells.Mutations in NOTCH1 play a central role in disruption of microenvironmental communication, potentially leading to cancer progression.Due to the dual, bi-directional signaling of NOTCH1, activatingmutations have been found in acute lymphoblastic leukemia and chroniclymphocytic leukemia, however loss of function mutations in NOTCH1 areprevalent in 11-15% of head and neck squamous cell carcinoma. NOTCH1mutations have also been found in 2% of glioblastomas, 1% of ovariancancers, 10% lung adenocarcinomas, 8% of squamous cell lung cancers and5% of breast cancers. Notch pathway-directed therapy approaches differdepending on whether the tumor harbors gain or loss of functionmutations, thus are classified as Notch pathway inhibitors oractivators, respectively. NPM1 NPM1 or nucleophosmin is a nucleolarphosphoprotein belonging to a family of nuclear chaperones withproliferative and growth-suppressive roles. In several hematologicalmalignancies, the NPM locus is lost or translocated, leading toexpression of oncogenic proteins. NPM1 is mutated in one-third ofpatients with adult acute myeloid leukemia (AML) leading to activationof downstream pathways including JAK/STAT, RAS/ERK, and PI3K. Althoughthere are few NPM-directed therapies currently being investigated,research shows AML tumor cells with mutant NPM are more sensitive tochemotherapeutic agents, including daunorubicin and camptothecin. NRASNRAS is an oncogene and a member of the (GTPase) ras family, whichincludes KRAS and HRAS. This biomarker has been detected in multiplecancers including melanoma (15%), colorectal cancer (4%), AML (10%) andbladder cancer (2%). Evidence suggests that an acquired mutation in NRASmay be associated with resistance to vemurafenib in melanoma patients.In colorectal cancer patients NRAS mutation is associated withresistance to EGFR-targeted monoclonal antibodies. Germline mutations inNRAS have been associated with Noonan syndrome, autoimmunelymphoproliferative syndrome and juvenile myelomonocytic leukemia. PBRM1This locus encodes a subunit of ATP-dependent chromatin-remodelingcomplexes. The encoded protein has been identified as in integralcomponent of complexes necessary for ligand-dependent transcriptionalactivation by nuclear hormone receptors. Mutations at this locus havebeen associated with primary clear cell renal cell carcinoma. PD-1 PD-1(programmed death 1) is a co-inhibitory receptor expressed on activatedT, B and NK cells, and tumor infiltrating lymphocytes (TIL). PD-1 is anegative regulator of the immune system and inhibits the proliferationand effector function of the lymphocytes after binding with its ligandsincluding PD-L1. PD-1/PD-L1 signaling pathway functions to attenuate orescape antitumor immunity by maintaining an immunosuppressive tumormicroenvironment. Studies show that the presence of PD-1 + TIL isassociated with a poor prognosis in various cancer types includinglymphoma and breast cancer. PD-L1 PD-L1 (programmed cell death ligand 1;also known as cluster of differentiation 274 (CD274) or B7 homolog 1(B7-H1)) is a glycoprotein expressed in various tumor types and isassociated with poor outcome. Upon binding to its receptor, PD-1, thePD-1/PD-L1 interaction functions to negatively regulate the immunesystem, attenuating antitumor immunity by maintaining animmunosuppressive tumor microenvironment. PD-L1 expression isupregulated in tumor cells through activation of common oncogenicpathways or exposure to inflammatory cytokines. Assessment of PD-L1offers information on patient prognosis and also represents a target forimmune manipulation in treatment of solid tumors. Clinical trials arecurrently recruiting patients with various tumor types testingimmunomodulatory agents. PDGFRA PDGFRA is the alpha-typeplatelet-derived growth factor receptor, a surface tyrosine kinasereceptor structurally homologous to c-KIT, which activates PIK3CA/AKT,RAS/MAPK and JAK/STAT signaling pathways. PDGFRA mutations are found in5-8% of patients with gastrointestinal stromal tumors (GIST) andincreases to 30% in KIT wildtype GIST. Germline mutations in PDGFRA havebeen associated with Familial gastrointestinal stromal tumors andHypereosinophillic Syndrome (HES). PGP P-glycoprotein (MDR1, ABCB1) isan ATP-dependent, transmembrane drug efflux pump with broad substratespecificity, which pumps antitumor drugs out of cells. Its expression isoften induced by chemotherapy drugs and is thought to be a majormechanism of chemotherapy resistance. Overexpression of p-gp isassociated with resistance to anthracylines (doxorubicin, epirubicin).P-gp remains the most important and dominant representative ofMulti-Drug Resistance phenotype and is correlated with disease state andresistant phenotype. PIK3CA (PI3K) PIK3CA (phosphoinositide-3-kinasecatalytic alpha polypeptide) encodes a protein in the PI3 kinasepathway. This pathway is an active target for drug development. PIK3CAsomatic mutations have been found in breast (26%), endometrial (23%),urinary tract (19%), colon (13%), and ovarian (11%) cancers. PIK3CA exon20 mutations have been associated with benefit from mTOR inhibitors(everolimus, temsirolimus). Evidence suggests that breast cancerpatients with activation of the PI3K pathway due to PTEN loss or PIK3CAmutation/amplification have a significantly shorter survival followingtrastuzumab treatment. PIK3CA mutated colorectal cancer patients areless likely to respond to EGFR targeted monoclonal antibody therapy.Somatic mosaic activating mutations in PIK3CA are said to cause CLOVESsyndrome. PMS2 This gene encodes the postmeiotic segregation increased 2(PMS2) protein involved in DNA mismatch repair. PMS2 forms a heterodimerwith MLH1 and, together, this complex interacts with other complexesbound to mismatched bases. Loss of PMS2 leads to mismatch repairdeficiency and microsatellite instability. Inactivating mutations inthis gene are associated with protein loss and hereditary Lynchsyndrome, the latter being linked with a lifetime risk for variousmalignancies, especially colorectal and endometrial cancer. PR Theprogesterone receptor (PR or PGR) is an intracellular steroid receptorthat specifically binds progesterone, an important hormone that fuelsbreast cancer growth. PR positivity in a tumor indicates that the tumoris more likely to be responsive to hormone therapy by anti-estrogens,aromatase inhibitors and progestogens. PTEN PTEN or phosphatase andtensin homolog is a tumor suppressor gene that prevents cells fromproliferating. PTEN is an important mediator in signaling downstream ofEGFR, and loss of PTEN gene function/expression due to gene mutations orallele loss is associated with reduced benefit to EGFR-targetedmonoclonal antibodies. Mutation in PTEN is found in 5-14% of colorectalcancer and 7% of breast cancer. PTEN mutation leads to loss of functionof the encoded phosphatase, and an upregulation of the PIK3CA/AKTpathway. Germline PTEN mutations associate with Cowden disease andBannayan-Riley-Ruvalcaba syndrome. These dominantly inherited disordersbelong to a family of hamartomatous polyposis syndromes which featuremultiple tumor-like growths (hamartomas) accompanied by an increasedrisk of breast carcinoma, follicular carcinoma of the thyroid, glioma,prostate and endometrial cancer. Trichilemmoma, a benign, multifocalneoplasm of the skin is also associated with PTEN germline mutations.PTPN11 PTPN11 or tyrosine-protein phosphatase non-receptor type 11 is aproto-oncogene that encodes a signaling molecule, Shp-2, which regulatesvarious cell functions like mitogenic activation and transcriptionregulation. PTPN11 gain-of-function somatic mutations have been found toinduce hyperactivation of the Akt and MAPK networks. Because of thishyperactivation, Ras effectors, such as Mek and PI3K, are potentialtargets for novel therapeutics in those with PTPN11 gain-of-functionmutations. PTPN11 somatic mutations are found in hematologic andlymphoid malignancies (8%), gastric (2%), colon (2%), ovarian (2%), andsoft tissue (2%) cancers. Germline mutations of PTPN11 are associatedwith Noonan syndrome, which itself is associated with juvenilemyelomonocytic leukemia (JMML). PTPN11 is also associated with LEOPARDsyndrome, which is associated with neuroblastoma and myeloid leukemia.RB1 RB1 or retinoblastoma-1 is a tumor suppressor gene whose proteinregulates the cell cycle by interacting with various transcriptionfactors, including the E2F family (which controls the expression ofgenes involved in the transition of cell cycle checkpoints). Besidesocular cancer, RB1 mutations have also been detected in othermalignancies, such as ovarian (10%), bladder (41%), prostate (8%),breast (6%), brain (6%), colon (5%), and renal (2%) cancers. RB1 status,along with other mitotic checkpoints, has been associated with theprognosis of GIST patients. Germline mutations of RB1 are associatedwith the pediatric tumor, retinoblastoma. Inherited retinoblastoma isusually bilateral. Studies indicate patients with a history ofretinoblastoma are at increased risk for secondary malignancies. RET RETor rearranged during transfection gene, located on chromosome 10,activates cell signaling pathways involved in proliferation and cellsurvival. RET mutations are found in 23-69% of sporadic medullarythyroid cancers (MTC), but RET fusions are common in papillary thyroidcancer, and more recently have been found in 1-2% of lungadenocarcinoma. Germline activating mutations of RET are associated withmultiple endocrine neoplasia type 2 (MEN2), which is characterized bythe presence of medullary thyroid carcinoma, bilateral pheochromocytoma,and primary hyperparathyroidism. Germline inactivating mutations of RETare associated with Hirschsprung's disease. ROS1 The proto-oncogene ROS1is a receptor tyrosine kinase of the insulin receptor family. The ligandand function of ROS1 are unknown. Dimerization of ROS1-fused proteinsresults in constitutive activation of the receptor kinase, leading tocell proliferation and survival. Clinical data show that ROS-rearrangedNSCLC patients have increased sensitivity and improved response to theMET/ALK/ROS inhibitor, crizotinib. RRM1 Ribonucleotide reductase subunitM1 (RRM1) is a component of the ribonucleotide reductase holoenzymeconsisting of M1 and M2 subunits. The ribonucleotide reductase is arate-limiting enzyme involved in the production of nucleotides requiredfor DNA synthesis. Gemcitabine is a deoxycitidine analogue whichinhibits ribonucleotide reductase activity. High RRM1 level isassociated with resistance to gemcitabine. SMAD4 SMAD4 or mothersagainst decapentaplegic homolog 4, is one of eight proteins in the SMADfamily, involved in multiple signaling pathways and are key modulatorsof the transcriptional responses to the transforming growth factor-ß(TGFß) receptor kinase complex. SMAD4 resides on chromosome 18q21, oneof the most frequently deleted chromosomal regions in colorectal cancer.Smad4 stabilizes Smad DNA-binding complexes and also recruitstranscriptional coactivators such as histone acetyltransferases toregulatory elements. Dysregulation of SMAD4 occurs late in tumordevelopment, and occurs through mutations of the MH1 domain whichinhibits the DNA-binding function, thus dysregulating TGFßR signaling.Mutated (inactivated) SMAD4 is found in 50% of pancreatic cancers and10-35% of colorectal cancers. Germline mutations in SMAD4 are associatedwith juvenile polyposis (JP) and combined syndrome of JP and hereditaryhemorrhagic teleangiectasia (JP-HHT). SMARCB1 SMARCB1 also known asSWI/SNF related, matrix associated, actin dependent regulator ofchromatin, subfamily b, member 1, is a tumor suppressor gene implicatedin cell growth and development. Loss of expression of SMARCB1 has beenobserved in tumors including epithelioid sarcoma, renal medullarycarcinoma, undifferentiated pediatric sarcomas, and a subset ofhepatoblastomas. Germline mutation in SMARCB1 causes about 20% of allrhabdoid tumors which makes it important for clinicians to facilitategenetic testing and refer families for genetic counseling. GermlineSMARCB1 mutations have also been identified as the pathogenic cause of asubset of schwannomas and meningiomas. SMO SMO (smoothened) is a Gprotein-coupled receptor which plays an important role in the Hedgehogsignaling pathway. It is a key regulator of cell growth anddifferentiation during development, and is important in epithelial andmesenchymal interaction in many tissues during embryogenesis.Dysregulation of the Hedgehog pathway is found in cancers includingbasal cell carcinomas (12%) and medulloblastoma (1%). A gain-of-functionmutation in SMO results in constitutive activation of hedgehog pathwaysignaling, contributing to the genesis of basal cell carcinoma. SMOmutations have been associated with the resistance to SMO antagonistGDC-0449 in medulloblastoma patients by blocking the binding to SMO. SMOmutation may also contribute partially to resistance to SMO antagonistLDE225 in BCC. Various clinical trials (on www.clinicaltrials.gov)investigating SMO antagonists may be available for SMO mutated patients.SPARC SPARC (secreted protein acidic and rich in cysteine) is acalcium-binding matricellular glycoprotein secreted by many types ofcells. Studies indicate SPARC over-expression improves the response tothe anticancer drug, nab-paclitaxel. The improved response is thought tobe related to SPARC's role in accumulating albumin and albumin-targetedagents within tumor tissue. STK11 STK11 also known as LKB1, is aserine/threonine kinase. It is thought to be a tumor suppressor genewhich acts by interacting with p53 and CDC42. It modulates the activityof AMP-activated protein kinase, causes inhibition of mTOR, regulatescell polarity, inhibits the cell cycle, and activates p53. Somaticmutations in this gene are associated with a history of smoking and KRASmutation in NSCLC patients. The frequency of STK11 mutation in lungadenocarcinomas ranges from 7%-30%. STK11 loss may play a role indevelopment of metastatic disease in lung cancer patients. Mutations ofthis gene also drive progression of HPV-induced dysplasia to invasive,cervical cancer and hence STK11 status may be exploited clinically topredict the likelihood of disease recurrence. Germline mutations inSTK11 are associated with Peutz-Jeghers syndrome which is characterizedby early onset hamartomatous gastro-intestinal polyps and increased riskof breast, colon, gastric and ovarian cancer. TLE3 TLE3 is a member ofthe transducin-like enhancer of split (TLE) family of proteins that havebeen implicated in tumorigenesis. It acts downstream of APC andbeta-catenin to repress transcription of a number of oncogenes, whichinfluence growth and microtubule stability. Studies indicate that TLE3expression is associated with response to taxane therapy. TOP2A TOPOIIAis an enzyme that alters the supercoiling of double-stranded DNA andallows chromosomal segregation into daughter cells. Due to its essentialrole in DNA synthesis and repair, and frequent overexpression in tumors,TOPOIIA is an ideal target for antineoplastic agents. Amplification ofTOPOIIA with or without HER2 co-amplification, as well as high proteinexpression of TOPOIIA, have been associated with benefit fromanthracycline based therapy. TOPO1 Topoisomerase I is an enzyme thatalters the supercoiling of double-stranded DNA. TOPOI acts bytransiently cutting one strand of the DNA to relax the coil and extendthe DNA molecule. Expression of TOPOI has been associated with responseto TOPOI inhibitors including irinotecan and topotecan. TP53 TP53, orp53, plays a central role in modulating response to cellular stressthrough transcriptional regulation of genes involved in cell-cyclearrest, DNA repair, apoptosis, and senescence. Inactivation of the p53pathway is essential for the formation of the majority of human tumors.Mutation in p53 (TP53) remains one of the most commonly describedgenetic events in human neoplasia, estimated to occur in 30-50% of allcancers. Generally, presence of a disruptive p53 mutation is associatedwith a poor prognosis in all types of cancers, and diminishedsensitivity to radiation and chemotherapy. In addition, various clinicaltrials (on www.clinicaltrials.gov) investigating agents which targetp53's downstream or upstream effectors may have clinical utilitydepending on the p53 status. For example, for p53 mutated patients, Chk1 inhibitors in advanced cancer and Wee1 inhibitors in ovarian cancerhave been investigated. For p53 wildtype patients with sarcoma, mdm2inhibitors have been investigated. Germline p53 mutations are associatedwith the Li-Fraumeni syndrome (LFS) which may lead to early-onset ofseveral forms of cancer currently known to occur in the syndrome,including sarcomas of the bone and soft tissues, carcinomas of thebreast and adrenal cortex (hereditary adrenocortical carcinoma), braintumors and acute leukemias. TS Thymidylate synthase (TS) is an enzymeinvolved in DNA synthesis that generates thymidine monophosphate (dTMP),which is subsequently phosphorylated to thymidine triphosphate for usein DNA synthesis and repair. Low levels of TS are predictive of responseto fluoropyrimidines and other folate analogues. TUBB3 Class IIIß-Tubulin (TUBB3) is part of a class of proteins that provide theframework for microtubules, major structural components of thecytoskeleton. Due to their importance in maintaining structuralintegrity of the cell, microtubules are ideal targets for anti-canceragents. Low expression of TUBB3 is associated with potential clinicalbenefit to taxane therapy. VHL VHL or von Hippel-Lindau gene encodes fortumor suppressor protein pVHL, which polyubiquitylates hypoxia-induciblefactor. Absence of pVHL causes stabilization of HIF and expression ofits target genes, many of which are important in regulatingangiogenesis, cell growth and cell survival. VHL somatic mutation hasbeen seen in 20-70% of patients with sporadic clear cell renal cellcarcinoma (ccRCC) and the mutation may imply a poor prognosis, adversepathological features, and increased tumor grade or lymph-nodeinvolvement. Renal cell cancer patients with a ‘loss of function’mutation in VHL show a higher response rate to therapy (bevacizumab orsorafenib) than is seen in patients with wild type VHL. Germlinemutations in VHL cause von Hippel-Lindau syndrome, associated withclear-cell renal-cell carcinomas, central nervous systemhemangioblastomas, pheochromocytomas and pancreatic tumors.

Table 7 shows exemplary MI molecular profiles for various tumorlineages. In the table, the lineage is shown in the column “Tumor Type.”The remaining columns show various biomarkers that can be assessed usingthe indicated methodology (i.e., immunohistochemistry (IHC), ISH orother techniques). One of skill will appreciate that similar methodologycan be employed as desired. For example, other suitable protein analysismethods can be used instead of IHC, other suitable nucleic acid analysismethods can be used instead of ISH (e.g., that assess copy number and/orrearrangements, translocations and the like), and other suitable nucleicacid analysis methods can be used instead of fragment analysis.Similarly, FISH and CISH are generally interchangeable and the choicemay be made based upon probe availability, resources, and the like.Table 8 presents a panel of genes that can be assessed as part of the MImolecular profiles using Next Generation Sequencing (NGS) analysis. Oneof skill will appreciate that other nucleic acid analysis methods can beused instead of NGS analysis, e.g., other sequencing, hybridization(e.g., microarray, Nanostring) and/or amplification (e.g., PCR based)methods.

In an embodiment, the invention provides a MI molecular profile forbladder cancer. The molecular profile may comprise IHC analysis of atleast one, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9, of ERCC1, Her2/Neu, PD-L1,PTEN, RRM1, TOP2A, TOPO1, TS, TUBB3; and/or ISH analysis of at leastTOP2A. The molecular profile may further comprise NGS analysis of atleast one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46, of ABL1, AKT1,ALK, APC, ATM, BRAF, BRCA1, BRCA2, CDH1, CSF1R, CTNNB1, EGFR, ERBB2(HER2), ERBB4 (HER4), FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS,HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT (cKIT), KRAS, MET(cMET), MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET,SMAD4, SMARCB1, SMO, STK11, TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile forbreast cancer. The molecular profile may comprise IHC analysis of atleast one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of AR, ER, ERCC1,Her2/Neu, PD-L1, PR, PTEN, RRM1, TLE3, TOPO1, TS; and/or ISH analysis ofat least one or two of Her2/Neu and TOP2A. The molecular profile mayfurther comprise NGS analysis of at least one, e.g., at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45 or 46, of ABL1, AKT1, ALK, APC, ATM, BRAF, BRCA1,BRCA2, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4 (HER4), FBXW7,FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3,KDR (VEGFR2), KIT (cKIT), KRAS, MET (cMET), MPL, NOTCH1, NPM1, NRAS,PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11,TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for acancer of unknown primary (CUP). The molecular profile may comprise IHCanalysis of at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12of AR, ER, ERCC1, Her2/Neu, PD-L1, PR, PTEN, RRM1, TOP2A, TOPO1, TS,TUBB3; and/or ISH analysis of at least Her2/Neu. The molecular profilemay further comprise NGS analysis of at least one, e.g., at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45 or 46, of ABL1, AKT1, ALK, APC, ATM, BRAF, BRCA1,BRCA2, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4 (HER4), FBXW7,FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3,KDR (VEGFR2), KIT (cKIT), KRAS, MET (cMET), MPL, NOTCH1, NPM1, NRAS,PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11,TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for acervical cancer. The molecular profile may comprise IHC analysis of atleast one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of ER, ERCC1,Her2/Neu, PD-L1, PR, PTEN, RRM1, TOP2A, TOPO1, TS, TUBB3; and/or ISHanalysis of at least one or two of Her2/Neu and TOP2A. The molecularprofile may further comprise NGS analysis of at least one, e.g., atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45 or 46, of ABL1, AKT1, ALK, APC, ATM,BRAF, BRCA1, BRCA2, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4(HER4), FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1,JAK2, JAK3, KDR (VEGFR2), KIT (cKIT), KRAS, MET (cMET), MPL, NOTCH1,NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO,STK11, TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for acolorectal cancer (CRC). The molecular profile may comprise IHC analysisof at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of ERCC1,HER2/Neu, MGMT, MLH1, MSH2, MSH6, PD-L1, PMS2, PTEN, TOPO1, TS; and/orISH analysis of at least one or two of Her2/Neu and TOP2A; and/or MSIanalysis. The molecular profile may further comprise NGS analysis of atleast one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46, of ABL1, AKT1,ALK, APC, ATM, BRAF, BRCA1, BRCA2, CDH1, CSF1R, CTNNB1, EGFR, ERBB2(HER2), ERBB4 (HER4), FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS,HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT (cKIT), KRAS, MET(cMET), MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET,SMAD4, SMARCB1, SMO, STK11, TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for anendometrial cancer. The molecular profile may comprise IHC analysis ofat least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15of ER, ERCC1, Her2/Neu, MLH1, MSH2, MSH6, PD-L1, PMS2, PR, PTEN, RRM1,TOP2A, TOPO1, TS, TUBB3; and/or ISH analysis of at least Her2/Neu;and/or MSI analysis. The molecular profile may further comprise NGSanalysis of at least one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or46, of ABL1, AKT1, ALK, APC, ATM, BRAF, BRCA1, BRCA2, CDH1, CSF1R,CTNNB1, EGFR, ERBB2 (HER2), ERBB4 (HER4), FBXW7, FGFR1, FGFR2, FLT3,GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT(cKIT), KRAS, MET (cMET), MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN,PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for agastric/esophageal cancer. The molecular profile may comprise IHCanalysis of at least one, e.g., 1, 2, 3, 4, 5, 6, 7 or 8 of ERCC1,Her2/Neu, PD-L1, PTEN, TOP2A, TOPO1, TS, TUBB3; and/or ISH analysis ofat least Her2/Neu. The molecular profile may further comprise NGSanalysis of at least one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or46, of ABL1, AKT1, ALK, APC, ATM, BRAF, BRCA1, BRCA2, CDH1, CSF1R,CTNNB1, EGFR, ERBB2 (HER2), ERBB4 (HER4), FBXW7, FGFR1, FGFR2, FLT3,GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT(cKIT), KRAS, MET (cMET), MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN,PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for agastrointestinal stromal tumor (GIST). The molecular profile maycomprise IHC analysis of at least one, e.g., 1, 2, 3 or 4 of ERCC1,Her2/Neu, PD-L1, PTEN; and/or ISH analysis of at least Her2/Neu. Themolecular profile may further comprise NGS analysis of at least one,e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46, of ABL1, AKT1, ALK,APC, ATM, BRAF, BRCA1, BRCA2, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2),ERBB4 (HER4), FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS,IDH1, JAK2, JAK3, KDR (VEGFR2), KIT (cKIT), KRAS, MET (cMET), MPL,NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4,SMARCB1, SMO, STK11, TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for aglioma. The molecular profile may comprise IHC analysis of at least one,e.g., 1, 2, 3, 4, 5, 6 or 7 of ERCC1, Her2/Neu, PD-L1, PTEN, TOPO1, TS,TUBB3; and/or ISH analysis of at least one or two of Her2/Neu and 1p19q;and/or fragment analysis of at least EGFR Variant III; and/or MGMTpromoter methylation analysis, e.g., by pyrosequencing. The molecularprofile may further comprise NGS analysis of at least one, e.g., atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45 or 46, of ABL1, AKT1, ALK, APC, ATM,BRAF, BRCA1, BRCA2, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4(HER4), FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1,JAK2, JAK3, KDR (VEGFR2), KIT (cKIT), KRAS, MET (cMET), MPL, NOTCH1,NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO,STK11, TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for ahead & neck cancer. The molecular profile may comprise IHC analysis ofat least one, e.g., 1, 2, 3, 4, 5, 6 or 7 of ERCC1, Her2/Neu, PD-L1,PTEN, RRM1, TS, TUBB3; and/or ISH analysis of at least Her2/Neu. Themolecular profile may further comprise NGS analysis of at least one,e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46, of ABL1, AKT1, ALK,APC, ATM, BRAF, BRCA1, BRCA2, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2),ERBB4 (HER4), FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS,IDH1, JAK2, JAK3, KDR (VEGFR2), KIT (cKIT), KRAS, MET (cMET), MPL,NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4,SMARCB1, SMO, STK11, TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for akidney cancer. The molecular profile may comprise IHC analysis of atleast one, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 of ERCC1, Her2/Neu, PD-L1,PTEN, RRM1, TOP2A, TOPO1, TS, TUBB3; and/or ISH analysis of at leastHer2/Neu. The molecular profile may further comprise NGS analysis of atleast one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46, of ABL1, AKT1,ALK, APC, ATM, BRAF, BRCA1, BRCA2, CDH1, CSF1R, CTNNB1, EGFR, ERBB2(HER2), ERBB4 (HER4), FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS,HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT (cKIT), KRAS, MET(cMET), MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET,SMAD4, SMARCB1, SMO, STK11, TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for amelanoma. The molecular profile may comprise IHC analysis of at leastone, e.g., 1, 2, 3, 4, 5, 6 or 7 of ERCC1, Her2/Neu, MGMT, PD-L1, PTEN,TS, TUBB3; and/or ISH analysis of at least Her2/Neu. The molecularprofile may further comprise NGS analysis of at least one, e.g., atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45 or 46, of ABL1, AKT1, ALK, APC, ATM,BRAF, BRCA1, BRCA2, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4(HER4), FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1,JAK2, JAK3, KDR (VEGFR2), KIT (cKIT), KRAS, MET (cMET), MPL, NOTCH1,NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO,STK11, TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for anon-small cell lung cancer (NSCLC). The molecular profile may compriseIHC analysis of at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 of ALK,ERCC1, Her2/Neu, PD-L1, PTEN, RRM1, TOPO1, TS, TUBB3; and/or ISHanalysis of at least one, e.g., 1, 2, 3 or 4 of cMET, EGFR, Her2/Neu andROS1. The molecular profile may further comprise NGS analysis of atleast one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46, of ABL1, AKT1,ALK, APC, ATM, BRAF, BRCA1, BRCA2, CDH1, CSF1R, CTNNB1, EGFR, ERBB2(HER2), ERBB4 (HER4), FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS,HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT (cKIT), KRAS, MET(cMET), MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET,SMAD4, SMARCB1, SMO, STK11, TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for anovarian cancer. The molecular profile may comprise IHC analysis of atleast one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of ER, ERCC1, Her2/Neu,PD-L1, PTEN, RRM1, TOP2A, TOPO1, TS, TUBB3; and/or ISH analysis of atleast Her2/Neu. The molecular profile may further comprise NGS analysisof at least one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46, ofABL1, AKT1, ALK, APC, ATM, BRAF, BRCA1, BRCA2, CDH1, CSF1R, CTNNB1,EGFR, ERBB2 (HER2), ERBB4 (HER4), FBXW7, FGFR1, FGFR2, FLT3, GNA11,GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT (cKIT),KRAS, MET (cMET), MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11,RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for apancreatic/hepatobiliary/cholangiocarcinoma cancer. The molecularprofile may comprise IHC analysis of at least one, e.g., 1, 2, 3, 4, 5,6, 7 or 8 of ERCC1, Her2/Neu, PD-L1, PTEN, RRM1, TOPO1, TS, TUBB3;and/or ISH analysis of at least Her2/Neu. The molecular profile mayfurther comprise NGS analysis of at least one, e.g., at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45 or 46, of ABL1, AKT1, ALK, APC, ATM, BRAF, BRCA1,BRCA2, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4 (HER4), FBXW7,FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3,KDR (VEGFR2), KIT (cKIT), KRAS, MET (cMET), MPL, NOTCH1, NPM1, NRAS,PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11,TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for aprostate cancer. The molecular profile may comprise IHC analysis of atleast one, e.g., 1, 2, 3, 4, 5, 6 or 7 of AR, ERCC1, Her2/Neu, PD-L1,PTEN, TOP2A, TUBB3; and/or ISH analysis of at least Her2/Neu. Themolecular profile may further comprise NGS analysis of at least one,e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46, of ABL1, AKT1, ALK,APC, ATM, BRAF, BRCA1, BRCA2, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2),ERBB4 (HER4), FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS,IDH1, JAK2, JAK3, KDR (VEGFR2), KIT (cKIT), KRAS, MET (cMET), MPL,NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4,SMARCB1, SMO, STK11, TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for asarcoma. The molecular profile may comprise IHC analysis of at leastone, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of ERCC1, Her2/Neu, MGMT,PD-L1, PTEN, RRM1, TOP2A, TOPO1, TS, TUBB3; and/or ISH analysis of atleast Her2/Neu. The molecular profile may further comprise NGS analysisof at least one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46, ofABL1, AKT1, ALK, APC, ATM, BRAF, BRCA1, BRCA2, CDH1, CSF1R, CTNNB1,EGFR, ERBB2 (HER2), ERBB4 (HER4), FBXW7, FGFR1, FGFR2, FLT3, GNA11,GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT (cKIT),KRAS, MET (cMET), MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11,RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile for athyroid cancer. The molecular profile may comprise IHC analysis of atleast one, e.g., 1, 2, 3, 4 or 5 of ERCC1, Her2/Neu, PD-L1, PTEN, TOP2A;and/or ISH analysis of at least Her2/Neu. The molecular profile mayfurther comprise NGS analysis of at least one, e.g., at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45 or 46, of ABL1, AKT1, ALK, APC, ATM, BRAF, BRCA1,BRCA2, CDH1, CSF1R, CTNNB1, EGFR, ERBB2 (HER2), ERBB4 (HER4), FBXW7,FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3,KDR (VEGFR2), KIT (cKIT), KRAS, MET (cMET), MPL, NOTCH1, NPM1, NRAS,PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11,TP53, and VHL.

In an embodiment, the invention provides a MI molecular profile forother tumors than those listed above. The molecular profile may compriseIHC analysis of at least one, e.g., 1, 2, 3, 4, 5, 6, 7 or 8 of ERCC1,Her2/Neu, PD-L1, PTEN, TOP2A, TOPO1, TS, TUBB3; and/or ISH analysis ofat least Her2/Neu. The molecular profile may further comprise NGSanalysis of at least one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or46, of ABL1, AKT1, ALK, APC, ATM, BRAF, BRCA1, BRCA2, CDH1, CSF1R,CTNNB1, EGFR, ERBB2 (HER2), ERBB4 (HER4), FBXW7, FGFR1, FGFR2, FLT3,GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT(cKIT), KRAS, MET (cMET), MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN,PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, and VHL.

Tables 7-8 provide various biomarkers that can be assessed for theindicated tumor lineages. Table 9 presents a view of associationsbetween the biomarkers assessed and various therapeutic agents. Suchassociations can be determined by correlating the biomarker assessmentresults with drug associations from sources such as the NCCN, literaturereports and clinical trials. The columns headed “Agent” providecandidate agents (e.g., drugs) or biomarker status to be included in thereport. In some cases, the agent comprises clinical trials that can bematched to a biomarker status. Where agents are indicated, theassociation of the agent with the indicated biomarker can included inthe MI report. In certain cases, multiple biomarkers are associated witha given agent or agents. For example, carboplatin, cisplatin,oxaliplatin are associated with BRCA1, BRCA2 and ERCC1. Platformabbreviations are as used throughout the application, e.g., IHC:immunohistochemistry; FISH: fluorescent in situ hybridication; CISH:colorimetric in situ hybridization; NGS: next generation sequencing;PCR: polymerase chain reaction. The candidate agents may comprise thoseundergoing clinical trials, as indicated. As will be evident to one ofskill, the same biomarkers in Table 7 can be assessed using theindicated methodology for both MI and MI Plus molecular profiling.

As described herein, the invention further provides a report comprisingresults of the molecular profiling and corresponding candidatetreatments that are identified as likely beneficial or likely notbeneficial.

TABLE 7 Molecular Profile and Report Parameters in situ Tumor TypeImmunohistochemistry (IHC) Hybridization (ISH) Other Bladder ERCC1,Her2/Neu, PD-L1, PTEN, TOP2A (CISH) RRM1, TOP2A, TOPO1, TS, TUBB3 BreastAR, ER, ERCC1, Her2/Neu, PD-L1, Her2/Neu, TOP2A PR, PTEN, RRM1, TLE3,TOPO1, (CISH) TS Cancer of Unknown AR, ER, ERCC1, Her2/Neu, PD-L1,Her2/Neu (CISH) Primary PR, PTEN, RRM1, TOP2A, TOPO1, TS, TUBB3 CervixER, ERCC1, Her2/Neu, PD-L1, PR, Her2/Neu, TOP2A PTEN, RRM1, TOP2A,TOPO1, TS, (CISH) TUBB3 Colorectal ERCC1, HER2/Neu, MGMT, MLH1,Her2/Neu, TOP2A MSI (Fragment MSH2, MSH6, PD-L1, PMS2, PTEN, (CISH)Analysis) TOPO1, TS Endometrial ER, ERCC1, Her2/Neu, MLH1, Her2/Neu(CISH) MSI (Fragment MSH2, MSH6, PD-L1, PMS2, PR, Analysis) PTEN, RRM1,TOP2A, TOPO1, TS, TUBB3 Gastric/Esophageal ERCC1, Her2/Neu, PD-L1, PTEN,Her2/Neu (CISH) TOP2A, TOPO1, TS, TUBB3 GIST ERCC1, Her2/Neu, PD-L1,PTEN Her2/Neu (CISH) Glioma ERCC1, Her2/Neu, PD-L1, PTEN, Her2/Neu(CISH); EGFR Variant III TOPO1, TS, TUBB3 1p19q (FISH) (FragmentAnalysis), MGMT Methylation (Pyro Sequencing) Head & Neck ERCC1,Her2/Neu, PD-L1, PTEN, Her2/Neu (CISH) RRM1, TS, TUBB3 Kidney ERCC1,Her2/Neu, PD-L1, PTEN, Her2/Neu (CISH) RRM1, TOP2A, TOPO1, TS, TUBB3Melanoma ERCC1, Her2/Neu, MGMT, PD-L1, Her2/Neu (CISH) PTEN, TS, TUBB3Non-Small Cell Lung ALK, ERCC1, Her2/Neu, PD-L1, cMET, EGFR, PTEN, RRM1,TOPO1, TS, TUBB3 Her2/Neu (CISH); ROS-1 (FISH) Ovarian ER, ERCC1,Her2/Neu, PD-L1, Her2/Neu (CISH) PTEN, RRM1, TOP2A, TOPO1, TS, TUBB3Pancreatic/ ERCC1, Her2/Neu, PD-L1, PTEN, Her2/Neu (CISH) Hepatobiliary/RRM1, TOPO1, TS, TUBB3 Cholangiocarcinoma Prostate AR, ERCC1, Her2/Neu,PD-L1, Her2/Neu (CISH) PTEN, TOP2A, TUBB3 Sarcoma ERCC1, Her2/Neu, MGMT,PD-L1, Her2/Neu (CISH) PTEN, RRM1, TOP2A, TOPO1, TS, TUBB3 ThyroidERCC1, Her2/Neu, PD-L1, PTEN, Her2/Neu (CISH) TOP2A Other Tumors ERCC1,Her2/Neu, PD-L1, PTEN, Her2/Neu (CISH) TOP2A, TOPO1, TS, TUBB3

TABLE 8 Next Generation Sequencing Markers ABL1 AKT1 ALK APC ATM BRCA1BRCA2 BRAF CDH1 CSF1R CTNNB1 EGFR ERBB2 (HER2) ERBB4 (HER4) FBXW7 FGFR1FGFR2 FLT3 GNA11 GNAQ GNAS HNF1A HRAS IDH1 JAK2 JAK3 KDR (VEGFR2) KIT(cKIT) KRAS MET (cMET) MPL NOTCH1 NPM1 NRAS PDGFRA PIK3CA PTEN PTPN11RB1 RET SMAD4 SMARCB1 SMO STK11 TP53 VHL

TABLE 9 Therapeutic Agent - Biomarker Associations Agent BiomarkerPlatform aspirin (assoc. in CRC only) PIK3CA NGS afatinib (assoc. inNSCLC only) EGFR NGS ERBB2 (HER2) NGS afatinib + cetuximab EGFR T790MNGS (combination assoc. in NSCLC only) cabozantinib (assoc. in NSCLCcMET NGS only) capecitabine, fluorouracil, TS IHC pemetrexedcarboplatin, cisplatin, BRCA1 NGS oxaliplatin BRCA2 NGS ERCC1 IHCceritinib ALK IHC cetuximab, BRAF NGS panitumumab (assoc. in CRC KRASNGS only) NRAS NGS PIK3CA NGS PTEN IHC cetuximab (assoc. in NSCLC EGFRCISH only) crizotinib ALK IHC cMET CISH, NGS ROS1 FISH dabrafenib,vemurafenib BRAF NGS dacarbazine, temozolomide MGMT IHC MGMT-MethylationPyrosequencing IDH1 (assoc. in High Grade Glioma only) NGS docetaxel,paclitaxel, nab- TLE3 IHC paclitaxel TUBB3 IHC doxorubicin, liposomal-HER2/Neu CISH doxorubicin, epirubicin TOP2A IHC CISH erlotinib,gefitinib EGFR NGS (assoc. in NSCLC only) KRAS NGS PIK3CA NGS cMET CISHPTEN IHC everolimus, temsirolimus ER (assoc. in Breast only) IHC PIK3CANGS gemcitabine RRM1 IHC hormone therapies AR IHC ER IHC PR IHC imatinibcKIT NGS PDGFRA NGS irinotecan TOPO1 IHC topotecan (excluding Breast,CRC, NSCLC) lapatinib, pertuzumab, T-DM1 HER2/Neu IHC; CISH lomustine,procarbazine, 1p19q FISH vincristine mitomycin-c BRCA1 NGS BRCA2nivolumab, pembrolizumab PD-L1 IHC (assoc. in Bladder, Kidney, Melanoma,NSCLC only) olaparib BRCA1 NGS (assoc. in Ovarian only) BRCA2osimertinib EGFR T790M NGS (assoc. in NSCLC only) palbociclib ER IHC(assoc. in Breast only) HER2/Neu IHC; CISH sunitinib (assoc. in GISTonly) cKIT NGS trametinib (assoc. in Melanoma BRAF NGS only) trastuzumabERBB2 (HER2) NGS (assoc. in NSCLC only) HER2/Neu IHC; CISH PTEN (assoc.in Breast only) IHC PIK3CA (assoc. in Breast only) NGS vandetanib RETNGS clinical trials EGFR PTEN IHC clinical trials EGFRvIII FragmentAnalysis clinical trials cMET CISH; NGS clinical trials MLH1, MSH2,MSH6, PMS2 IHC MSI Fragment Analysis clinical trials ABL1, AKT1, ALK,APC, ATM, CSF1R, NGS CTNNB1, EGFR, ERBB2 (Her2), FGFR1, FGFR2, FLT3,GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS, MPL, NOTCH1,NRAS, PTEN, SMO, TP53, VHL

With regard to MI) molecular profiles, cetuximab/panitumumab,vemurafenib/dabrafenib, and trametinib may be reported in combinationfor CRC. Hormone therapies may include: tamoxifen, toremifene,fulvestrant, letrozole, anastrozole, exemestane, megestrol acetate,leuprolide, goserelin, bicalutamide, flutamide, abiraterone,enzalutamide, triptorelin, abarelix, degarelix.

The biomarker-treatment associations can follow certain rules. The rulescomprise a predicted likelihood of benefit or lack of benefit of acertain treatment for the cancer given an assessment of one or morebiomarker. Exemplary associations/rules are presented in Table 10.Additional biomarker-drug associations can be found in the followingInternational Patent Applications, each of which is incorporated hereinby reference in its entirety: PCT/US2007/69286, filed May 18, 2007;PCT/US2009/60630, filed Oct. 14, 2009; PCT/2010/000407, filed Feb. 11,2010; PCT/US12/41393, filed Jun. 7, 2012; PCT/US2013/073184, filed Dec.4, 2013; PCT/US2010/54366, filed Oct. 27, 2010; PCT/US11/67527, filedDec. 28, 2011; and PCT/US15/13618, filed Jan. 29, 2015. In Table 10, theclass of drug and illustrative drugs of the indicated class areindicated in the columns “Class of Drugs” and “Drugs,” respectively. Thecolumns headed “Biomarker Result” illustrate illustrative methods ofprofiling the indicated biomarkers, wherein the results are generallytrue (“T”) or false (“F”), “Any,” or “No Data.” The data can also belabeled “Equivocal,” “Equivocal Low,” or “Equivocal High,” e.g., for IHCwhere the observed expression level is near or at the threshold set todetermine whether a protein is under-expressed, over-expressed, orexpressed at normal levels. For mutations, in some cases a particularmutation (e.g., BRAF V600E or V600K) or region/mutational hotspot iscalled out (e.g., c-KIT exon11 or exon13). In some cases, a particularmutation is called out from others in the “Biomarker Result.” Forexample, in the case of cKIT, the V654A mutation or mutations in exon14, exon 17, or exon 18 are called out in the rules for the tyrosinekinase inhibitor (“TKI”) imatinib. Similarly, in the case of PDGFRAmutations, the PDGFRA D842V mutation may be called out in the tablesapart from other PDGFRA mutations. One of skill will appreciate thatalternative methods can be used to analyze the biomarkers asappropriate. For example, sequencing analysis performed by NextGeneration methodology could also be performed by Sanger sequencing orother forms of sequence analysis method such as those described hereinor known in the art that yield similar biological information (e.g., anexpression or mutation status). The biomarker results combine to predicta benefit or lack of benefit from treatment with the indicated candidatedrugs. Abbreviations used in Table 10 include: tyrosine kinase inhibitor(“TKI”); Sequencing (“Seq.”); Indeterminate (“Indet.”); True (“T”);False (“F”).

As an example in Table 10, consider that PIK3CA exon20 is mutated asdetermined by sequencing (PIK3CA Mutated|exon20=T), then the mTORinhibitor agents everolimus and/or temsirolimus are predicted to havetreatment benefit (Overall Benefit=T). However, if PIK3CA exon20mutation is determined to be false (“F”) or is not determined (“NoData”), then the overall benefit of the mTOR inhibitors isindeterminate. As another example in Table 10, consider that the sampleis determined to be ER positive by IHC. In such case, overall benefitfrom the hormonal agents leuprolide and/or megestrol acetate is expectedto be likely (i.e., true or “T”). These results are independent of thestatus of PR as also determined by IHC. If ER is determined to not beoverexpressed (i.e., false “F”) or no data is available, and PR isdetermined to be positive by IHC, then overall benefit from theindicated hormonal agents such as leuprolide and megestrol acetate isalso expected to be likely (i.e., true or “T”). If neither ER nor PR areexpressed (i.e., ER Positive=false (“F”) and PR Positive=false (“F”)),then overall benefit from the hormonal agents leuprolide and/ormegestrol acetate is expected to be not likely (i.e., false or “F”). Theexpected overall benefit from the hormonal agents is indeterminate(i.e., “Indet.”) in either of the following situations: 1) ER is notexpressed or data is unavailable (i.e., ER Positive=“No Data”) and datais unavailable for PR (i.e., PR Positive=“No Data”); or 2) data isunavailable for ER (i.e., ER Positive=“No Data”) and PR is not expressed(i.e., PR Positive=“F”).

In addition to the columns in the tables above, Table 10 provides apredicted benefit level and an evidence level, and list of referencesfor each biomarker-drug association rule in the table. The benefit levelis ranked from 1-5, wherein the levels indicate the predicted strengthof the biomarker-drug association based on the indicated evidence. Allrelevant published studies were evaluated using the U.S. PreventiveServices Task Force (“USPSTF”) grading scheme for study design andvalidity. See, e.g.,www.uspreventiveservicestaskforce.org/uspstf/grades.htm. The benefitlevel in the table (“Bene. Level”) corresponds to the following:

1: Expected benefit.

2: Expected reduced benefit.

3: Expected lack of benefit.

4: No data is available.

5: Data is available but no expected benefit or lack of benefit reportedbecause the biomarker in this case is the not principal driver of thatspecific rule.

The evidence level in the table (“Evid. Level”) corresponds to thefollowing:

1: Very high level of evidence. For example, the treatment comprises thestandard of care.

2: High level of evidence but perhaps insufficient to be considered forstandard of care.

3: Weaker evidence—fewer publications or clinical studies, or perhapssome controversial evidence.

Abbreviations used in Table 10 include: Bene. (Benefit); Evid.(Evidence); Indet. (Indeterminate); Equiv. (Equivocal); Seq.(Sequencing). In the column “Drugs,” under the section for Taxanes, thefollowing abbreviations are used: PDN (paclitaxel, docetaxel,nab-paclitaxel) and N (nab-paclitaxel).

The column “Partial Report Overall Benefit” in Table 10 is to make drugassociation in a preliminary molecular profiling report if all thebiomarker assessment results are not ready. For example, a preliminaryreport may be produced when requested by the treating physician.Interpretation of benefit of lack of benefit of the various drugs may bemore cautious in these scenarios to avoid potential change in drugassociation from benefit or lack of benefit or vice versa between thepreliminary report and a final report that is produced when allbiomarker results become available. Hence there are some indeterminatescenarios.

TABLE 10 Solid Tumor Drug - Biomarker Associations Biomarker Bene. Evid.Ref. Biomarker Bene. Evid. Ref. Biomarker Bene. Evid. Class of DrugsDrugs Result Level Level No. Result Level Level No. Result Level LevelRRM1 Negative Bene. Evid. Antimetabolites gemcitabine (IHC) Level Level1 T 1 2 F 3 2 No Data 4 fluorouracil, TS capecitabine, Negative Bene.Evid. Antimetabolites pemetrexed (IHC) Level Level 2 T 1 2 F 3 2 No Data4 TOPO1 Topo1 irinotecan, Positive Bene. Evid. inhibitors topotecan(IHC) Level Level 3 T 1 2 F 3 2 No Data 4 MGMT Alkylating temozolomide,Negative Bene. Evid. agents dacarbazine (IHC) Level Level 4 T 1 2 F 3 2No Data 4 bicalutamide, flutamide, AR abiraterone, Positive Bene. Evid.Anti-androgens enzalutamide (IHC) Level Level 5 T 1 2 F 3 2 No Data 4tamoxifen, toremifene, fulvestrant, letrozole, anastrozole, exemestane,ER PR Hormonal megestrol Positive Bene. Evid. Positive Bene. Evid.Agents acetate (IHC) Level Level 6 (IHC) Level Level 7 T 1 1 T 1 1 T 1 1F 2 1 T 1 1 No Data 4 F 2 1 T 1 1 F 3 1 F 3 1 F 3 1 No Data 4 No Data 4T 1 1 No Data 4 F 3 1 No Data 4 No Data 4 HER2 HER2 Positive Bene. Evid.Amplified Bene. Evid. TKI lapatinib (IHC) Level Level 8 (ISH) LevelLevel 9 T 1 1 T 1 1 T 1 1 F 5 T 1 1 Equiv. High 1 1 T 1 1 Equiv. Low 5 T1 1 No Data 4 F 5 T 1 1 F 3 1 F 3 1 F 5 Equiv. High 1 1 F 3 1 Equiv. Low3 1 F 3 1 No Data 4 Equiv. 5 T 1 1 Equiv. 5 F 3 1 Equiv. 5 Equiv. High 11 Equiv. 5 Equiv. Low 3 1 Equiv. 5 No Data 4 No Data 4 T 1 1 No Data 4 F3 1 No Data 4 Equiv. High 1 1 No Data 4 Equiv. Low 3 1 No Data 4 No Data4 trastuzumab, pertuzumab, Monoclonal ado- antibodies trastuzumab HER2HER2 (Her2- emtansine Positive Bene. Evid. Amplified Bene. Evid.Targeted) (T-DM1) (IHC) Level Level 10 (ISH) Level Level 11 T 1 1 T 1 1T 1 1 F 5 T 1 1 Equiv. low 5 T 1 1 Equiv. high 1 1 T 1 1 No Data 4 F 5 T1 1 F 3 1 F 3 1 F 3 1 Equiv. low 3 1 F 5 Equiv. high 1 1 F 3 1 No Data 4Equiv. 5 T 1 1 Equiv. 5 F 3 1 Equiv. 5 Equiv. low 3 1 Equiv. 5 Equiv.high 1 1 Equiv. 5 No Data 4 No Data 4 T 1 1 No Data 4 F 3 1 No Data 4Equiv. low 3 1 No Data 4 Equiv. high 1 1 No Data 4 No Data 4doxorubicin, Anthracyclines liposomal- TOP2A Her2 TOP2A and relateddoxorubicin, Amplified Bene. Evid. Amplified Bene. Evid. Positive Bene.Evid. substances epirubicin (ISH) Level Level 12 (ISH) Level Level 13(IHC) Level Level T 1 1 T 1 1 T 1 2 T 1 1 T 1 1 T 1 2 T 1 1 T 1 1 T 1 2T 1 1 T 1 1 F 2 2 T 1 1 T 1 1 F 2 2 T 1 1 T 1 1 F 2 2 T 1 1 T 1 1 NoData 4 T 1 1 T 1 1 No Data 4 T 1 1 T 1 1 No Data 4 T 1 1 F 2 2 T 1 2 T 11 F 2 2 T 1 2 T 1 1 F 2 2 T 1 2 T 1 1 F 2 1 F 2 2 T 1 1 F 2 1 F 2 2 T 11 F 2 1 F 2 2 T 1 1 F 2 1 No Data 4 T 1 1 F 2 1 No Data 4 T 1 1 F 2 1 NoData 4 T 1 1 No Data 4 T 1 2 T 1 1 No Data 4 T 1 2 T 1 1 No Data 4 T 1 2T 1 1 No Data 4 F 2 2 T 1 1 No Data 4 F 2 2 T 1 1 No Data 4 F 2 2 T 1 1No Data 4 No Data 4 T 1 1 No Data 4 No Data 4 T 1 1 No Data 4 No Data 4T 1 1 Equiv. high 1 1 T 1 2 T 1 1 Equiv. high 1 1 T 1 2 T 1 1 Equiv.high 1 1 T 1 2 T 1 1 Equiv. high 1 1 F 2 2 T 1 1 Equiv. high 1 1 F 2 2 T1 1 Equiv. high 1 1 F 2 2 T 1 1 Equiv. high 1 1 No Data 4 T 1 1 Equiv.high 1 1 No Data 4 T 1 1 Equiv. high 1 1 No Data 4 T 1 1 Equiv. low 2 2T 1 2 T 1 1 Equiv. low 2 2 T 1 2 T 1 1 Equiv. low 2 2 T 1 2 T 1 1 Equiv.low 2 1 F 2 2 T 1 1 Equiv. low 2 1 F 2 2 T 1 1 Equiv. low 2 1 F 2 2 T 11 Equiv. low 2 1 No Data 4 T 1 1 Equiv. low 2 1 No Data 4 T 1 1 Equiv.low 2 1 No Data 4 F 2 2 T 1 1 T 1 2 F 2 2 T 1 1 T 1 2 F 2 2 T 1 1 T 1 2F 2 1 T 1 1 F 2 2 F 2 1 T 1 1 F 2 2 F 2 1 T 1 1 F 2 2 F 2 1 T 1 1 NoData 4 F 2 1 T 1 1 No Data 4 F 2 1 T 1 1 No Data 4 F 2 2 F 2 2 T 1 2 F 22 F 2 2 T 1 2 F 2 2 F 2 2 T 1 2 F 3 1 F 3 1 F 3 2 F 3 1 F 3 1 F 3 2 F 31 F 3 1 F 3 2 F 3 1 F 3 1 No Data 4 F 3 1 F 3 1 No Data 4 F 3 1 F 3 1 NoData 4 F 2 2 No Data 4 T 1 2 F 2 2 No Data 4 T 1 2 F 2 2 No Data 4 T 1 2F 3 1 No Data 4 F 3 2 F 3 1 No Data 4 F 3 2 F 3 1 No Data 4 F 3 2 F 3 1No Data 4 No Data 4 F 3 1 No Data 4 No Data 4 F 3 1 No Data 4 No Data 4F 2 2 Equiv. high 1 1 T 1 2 F 2 2 Equiv. high 1 1 T 1 2 F 2 2 Equiv.high 1 1 T 1 2 F 2 1 Equiv. high 1 1 F 2 2 F 2 1 Equiv. high 1 1 F 2 2 F2 1 Equiv. high 1 1 F 2 2 F 2 1 Equiv. high 1 1 No Data 4 F 2 1 Equiv.high 1 1 No Data 4 F 2 1 Equiv. high 1 1 No Data 4 F 2 2 Equiv. low 2 2T 1 2 F 2 2 Equiv. low 2 2 T 1 2 F 2 2 Equiv. low 2 2 T 1 2 F 3 1 Equiv.low 3 1 F 3 2 F 3 1 Equiv. low 3 1 F 3 2 F 3 1 Equiv. low 3 1 F 3 2 F 31 Equiv. low 3 1 No Data 4 F 3 1 Equiv. low 3 1 No Data 4 F 3 1 Equiv.low 3 1 No Data 4 No Data 4 T 1 1 T 1 2 No Data 4 T 1 1 T 1 2 No Data 4T 1 1 T 1 2 No Data 4 T 1 1 F 2 2 No Data 4 T 1 1 F 2 2 No Data 4 T 1 1F 2 2 No Data 4 T 1 1 No Data 4 No Data 4 T 1 1 No Data 4 No Data 4 T 11 No Data 4 No Data 4 F 2 2 T 1 2 No Data 4 F 2 2 T 1 2 No Data 4 F 2 2T 1 2 No Data 4 F 3 1 F 3 2 No Data 4 F 3 1 F 3 2 No Data 4 F 3 1 F 3 2No Data 4 F 3 1 No Data 4 No Data 4 F 3 1 No Data 4 No Data 4 F 3 1 NoData 4 No Data 4 No Data 4 T 1 2 No Data 4 No Data 4 T 1 2 No Data 4 NoData 4 T 1 2 No Data 4 No Data 4 F 3 2 No Data 4 No Data 4 F 3 2 No Data4 No Data 4 F 3 2 No Data 4 No Data 4 No Data 4 No Data 4 No Data 4 NoData 4 No Data 4 No Data 4 No Data 4 No Data 4 Equiv. high 1 1 T 1 2 NoData 4 Equiv. high 1 1 T 1 2 No Data 4 Equiv. high 1 1 T 1 2 No Data 4Equiv. high 1 1 F 2 2 No Data 4 Equiv. high 1 1 F 2 2 No Data 4 Equiv.high 1 1 F 2 2 No Data 4 Equiv. high 1 1 No Data 4 No Data 4 Equiv. high1 1 No Data 4 No Data 4 Equiv. high 1 1 No Data 4 No Data 4 Equiv. low 22 T 1 2 No Data 4 Equiv. low 2 2 T 1 2 No Data 4 Equiv. low 2 2 T 1 2 NoData 4 Equiv. low 3 1 F 3 2 No Data 4 Equiv. low 3 1 F 3 2 No Data 4Equiv. low 3 1 F 3 2 No Data 4 Equiv. low 3 1 No Data 4 No Data 4 Equiv.low 3 1 No Data 4 No Data 4 Equiv. low 3 1 No Data 4 PDGFRA c-KIT exon12 | exon11 | exon 14 | exon13 Bene. Evid. exon 18 Bene. Evid. TKIimatinib (Seq.) Level Level 16 (Seq.) Level Level 17 T 1 2 T 1 2 T 1 2 F5 T 2 2 D842V 3 2 T 1 2 No Data 4 F 2 2 T 1 2 F 3 2 F 3 2 F 3 2 D842V 32 F 3 2 No Data 4 V654A 3 2 T 2 2 V654A 3 2 F 3 2 V654A 3 2 D842V 3 2V654A 3 2 No Data 4 exon 14 5 T 1 2 exon 14 5 F 3 2 exon 14 5 D842V 3 2exon 14 5 No Data 4 exon 17 or 18 5 T 1 2 exon 17 or 18 5 F 3 2 exon 17or 18 5 D842V 3 2 exon 17 or 18 5 No Data 4 No Data 4 T 1 2 No Data 4 F3 2 No Data 4 D842V 3 2 No Data 4 No Data 4 ALK ROS1 Positive Bene.Evid. Positive Bene. Evid. TKI (crizotinib) crizotinib (ISH) Level Level18 (ISH) Level Level 19 T 1 2 T 1 2 F 5 T 1 2 No Data 4 T 1 2 T 1 2 F 5F 3 2 F 3 2 No Data 4 F 3 2 T 1 2 No Data 4 F 3 2 No Data 4 No Data 4 NoData 4 PIK3CA mTOR everolimus, exon20 Bene. Evid. inhibitorstemsirolimus (Seq.) Level Level 20 T 1 2 F 3 2 No Data 4 RET TKI (RET-Mutated Bene. Evid. targeted) vandetanib (Seq.) Level Level 21 T 1 1 F 5No Data 4 cisplatin, BRCA1 BRCA2 Platinum carboplatin, mutated Bene.Evid. mutated Bene. Evid. compounds oxaliplatin (Seq.) Level Level 22(Seq.) Level Level 23 T 1 2 T 1 2 T 1 2 F 5 T 1 2 No Data 4 F 5 T 1 2 F3 2 F 3 2 F 3 2 No Data 4 No Data 4 T 1 2 No Data 4 F 3 2 No Data 4 NoData 4 goserelin, leuprolide, triptorelin, AR ER GnRH agonists,abarelix, Positive Bene. Evid. Positive PR Bene. Evid. antagonistsdegarelix (IO) Level Level 24 (IHC) 25 Positive Level Level T 1 2 T 1 2T 1 2 T 1 2 T 1 2 F 2 2 T 1 2 T 1 2 No Data 4 T 1 2 F 2 2 T 1 2 T 1 2 F2 2 F 2 2 T 1 2 F 2 2 No Data 4 T 1 2 No Data 4 T 1 2 T 1 2 No Data 4 F2 2 T 1 2 No Data 4 No Data 4 F 2 2 T 1 2 T 1 2 F 2 2 T 1 2 F 2 2 F 2 2T 1 2 No Data 4 F 2 2 F 2 2 T 1 2 F 3 2 F 3 2 F 3 2 F 3 2 F 3 2 No Data4 F 2 2 No Data 4 T 1 2 F 3 2 No Data 4 F 3 2 F 3 2 No Data 4 No Data 4No Data 4 T 1 2 T 1 2 No Data 4 T 1 2 F 2 2 No Data 4 T 1 2 No Data 4 NoData 4 F 2 2 T 1 2 No Data 4 F 3 2 F 3 2 No Data 4 F 3 2 No Data 4 NoData 4 No Data 4 T 1 2 No Data 4 No Data 4 F 3 2 No Data 4 No Data 4 NoData 4 TLE3 TUBB3 PGP docetaxel, Positive Bene. Evid. Positive Bene.Evid. Positive Bene. Evid. Taxanes paclitaxel (IHC) Level Level 26 (IHC)Level Level 27 (IHC) Level Level T 1 2 T 2 2 T 2 3 T 1 2 F 1 2 T 2 3 T 12 No Data 4 T 2 3 F 3 2 T 3 2 T 3 3 F 2 2 F 1 2 T 2 3 F 3 2 No Data 4 T3 3 No Data 4 T 3 2 T 3 3 No Data 4 F 1 2 T 2 3 No Data 4 No Data 4 T 33 T 1 2 T 2 2 F 1 3 T 1 2 F 1 2 F 1 3 T 1 2 No Data 4 F 1 3 F 3 2 T 3 2F 1 3 F 2 2 F 1 2 F 1 3 F 3 2 No Data 4 F 1 3 No Data 4 T 3 2 F 1 3 NoData 4 F 1 2 F 1 3 No Data 4 No Data 4 F 1 3 T 1 2 T 2 2 No Data 4 T 1 2F 1 2 No Data 4 T 1 2 No Data 4 No Data 4 F 3 2 T 3 2 No Data 4 F 2 2 F1 2 No Data 4 F 3 2 No Data 4 No Data 4 No Data 4 T 3 2 No Data 4 NoData 4 F 1 2 No Data 4 No Data 4 No Data 4 No Data 4 SPARC SPARC Taxanes(nab- IHC Mono Bene. Evid. IHC Poly Bene. Evid. paclitaxel)nab-paclitaxel Pos. Level Level 29 Pos. Level Level 29 T 1 2 T 1 2 T 1 2F 2 2 T 1 2 No Data 4 F 2 2 T 1 2 F 3 2 F 3 2 F 3 2 No Data 4 No Data 4T 1 2 No Data 4 F 3 2 No Data 4 No Data 4 BRAF vemurafenib, V600Edabrafenib, (PCR or Bene. Evid. TKI trametinib seq.) Level Level 30 T 12 F 3 2 No Data 4 ALK Bene. Evid. TKI ceritinib Positive Level Level 31T 1 2 F 3 2 No Data 4 Partial Report Ref. Biomarker Bene. Evid. Ref.Biomarker Bene. Evid. Ref. Overall Overall Class of Drugs Drugs No.Result Level Level No. Result Level Level No. Bene. Bene. Partial ReportOverall Overall Antimetabolites gemcitabine Bene. Bene. T T F F Indet.Indet. Partial fluorouracil, Report capecitabine, Overall OverallAntimetabolites pemetrexed Bene. Bene. T T F F Indet. Indet. PartialReport Topo1 irinotecan, Overall Overall inhibitors topotecan Bene.Bene. T T F F Indet. Indet. Partial Report Alkylating temozolomide,Overall Overall agents dacarbazine Bene. Bene. T T F F Indet. Indet.bicalutamide, Partial flutamide, Report abiraterone, Overall OverallAnti-androgens enzalutamide Bene. Bene. T T F F Indet. Indet. tamoxifen,toremifene, fulvestrant, letrozole, anastrozole, Partial exemestane,Report Hormonal megestrol Overall Overall Agents acetate Bene. Bene. T TT T T T T T F F Indet. Indet. T T Indet. Indet. Indet. Indet. PartialReport Overall Overall TKI lapatinib Bene. Bene. T T T T T T T T T T T TF F T T F F Indet. Indet. T T F F T T F F Indet. Indet. T T Indet.Indet. T T Indet. Indet. Indet. Indet. trastuzumab, pertuzumab,Monoclonal ado- Partial antibodies trastuzumab Report (Her2- emtansineOverall Overall Targeted) (T-DM1) Bene. Bene. T T T T T T T T T T T T FF F F T T Indet. Indet. T T F F F F T T Indet. Indet. T T Indet. Indet.Indet. Indet. T T Indet. Indet. doxorubicin, Partial Anthracyclinesliposomal- PGP Report and related doxorubicin, Positive Bene. Evid.Overall Overall substances epirubicin 14 (IHC) Level Level 15 Bene.Bene. T 2 2 T T F 1 2 T T No Data 4 T T T 2 2 T T F 1 2 T T No Data 4 TT T 2 2 T T F 1 2 T T No Data 4 T T T 2 2 T T F 1 2 T T No Data 4 T T T2 2 T T F 1 2 T T No Data 4 T T T 2 2 T T F 1 2 T T No Data 4 T T T 2 2T T F 1 2 T T No Data 4 T T T 2 2 T T F 1 2 T T No Data 4 T T T 2 2 T TF 1 2 T T No Data 4 T T T 2 2 T T F 1 2 T T No Data 4 T T T 2 2 T T F 12 T T No Data 4 T T T 2 2 T T F 1 2 T T No Data 4 T T T 2 2 T T F 1 2 TT No Data 4 T T T 2 2 T T F 1 2 T T No Data 4 T T T 2 2 T T F 1 2 T T NoData 4 T T T 2 2 T T F 1 2 T T No Data 4 T T T 2 2 T T F 1 2 T T No Data4 T T T 2 2 T T F 1 2 T T No Data 4 T T T 2 2 T T F 1 2 T T No Data 4 TT T 3 2 F F F 2 2 F F No Data 4 F F T 3 2 F Indet. F 2 2 F Indet. NoData 4 F Indet. T 2 2 T T F 1 2 T T No Data 4 T T T 3 2 F Indet. F 2 2 FIndet. No Data 4 F Indet. T 3 2 F Indet. F 2 2 F Indet. No Data 4 FIndet. T 2 2 T T F 1 2 T T No Data 4 T T T 2 2 T T F 1 2 T T No Data 4 TT T 2 2 T T F 1 2 T T No Data 4 T T T 2 2 T T F 1 2 T T No Data 4 T T T3 2 F F F 2 2 F F No Data 4 F F T 3 2 F Indet. F 2 2 F Indet. No Data 4F Indet. T 2 2 T T F 1 2 T T No Data 4 T T T 2 2 T T F 1 2 T T No Data 4T T T 2 2 T T F 1 2 T T No Data 4 T T T 2 2 T T F 1 2 T T No Data 4 T TT 3 2 F Indet. F 2 2 F Indet. No Data 4 F Indet. T 3 2 F Indet. F 2 2 FIndet. No Data 4 F Indet. T 2 2 T T F 1 2 T T No Data 4 T T T 3 2 FIndet. F 2 2 F Indet. No Data 4 F Indet. T 3 2 F Indet. F 1 2 T Indet.No Data 4 Indet. Indet. T 2 2 T T F 1 2 T T No Data 4 T T T 2 2 T T F 12 T T No Data 4 T T T 2 2 T T F 1 2 T T No Data 4 T T T 2 2 T T F 1 2 TT No Data 4 T T T 3 2 F Indet. F 2 2 F Indet. No Data 4 F Indet. T 3 2 FIndet. F 2 2 F Indet. No Data 4 F Indet. Partial Report Overall OverallTKI imatinib Bene. Bene. T T T T F F T Indet. T T Indet. Indet. F FIndet. Indet. F F F F F F F F T T Indet. Indet. F F Indet. Indet. T TIndet. Indet. F F Indet. Indet. T Indet. Indet. Indet. F F Indet. Indet.Partial Report Overall Overall TKI (crizotinib) crizotinib Bene. Bene. TT T T T T T T F F Indet. Indet. T T F Indet. Indet. Indet. PartialReport mTOR everolimus, Overall Overall inhibitors temsirolimus Bene.Bene. T T Indet. Indet. Indet. Indet. Partial Report TKI (RET- OverallOverall targeted) vandetanib Bene. Bene. T T Indet. Indet. Indet. Indet.Partial cisplatin, Report Platinum carboplatin, Overall Overallcompounds oxaliplatin Bene. Bene. T T T T T T T T Indet. Indet. Indet.Indet. T T Indet. Indet. Indet. Indet. goserelin, leuprolide, Partialtriptorelin, Report GnRH agonists, abarelix, Overall Overall antagonistsdegarelix 25 Bene. Bene. T T T T T T T T T T T T T T T T T T T T T T T TT T F F F Indet. T T F Indet. F Indet. T T T Indet. T Indet. T T FIndet. F Indet. T T F Indet. Indet. Indet. Partial Report docetaxel,Overall Overall Taxanes paclitaxel 28 Bene. Bene. T T T T T T F F T T FIndet. F Indet. T T Indet. Indet. T T T T T T F F T T F Indet. F Indet.T T Indet. Indet. T T T T T T F F T T F Indet. F Indet. T T Indet.Indet. Partial Report Taxanes (nab- Overall Overall paclitaxel)nab-paclitaxel Bene. Bene. T T T T T T T T Indet. Indet. Indet. Indet. TT Indet. Indet. Indet. Indet. Partial vemurafenib, Report dabrafenib,Overall Overall TKI trametinib Bene. Bene. T T F F Indet. Indet. PartialReport Overall Overall TKI ceritinib Bene. Bene. T T F F Indet. Indet.

Table 11 contains the references used to predict benefit level andprovide an evidence level as shown in Table 10 above. The “Ref. No.”column in Table 11 corresponds to the “Ref. No.” columns in Table 10.Specifically, the reference numbers in Table 10 include those referencesindicated in Table 11.

TABLE 11 References for Solid Tumor Molecular Profile Ref. No.References 1 Gong, W., J. Dong, et. al. (2012). “RRM1 expression andclinical outcome of gemcitabine- containing chemotherapy for advancednon-small-cell lung cancer: A meta-analysis.” Lung Cancer. 75: 374-380.2 Qiu, L. X., M. H. Zheng, et. al. (2008). “Predictive value ofthymidylate synthase expression in advanced colorectal cancer patientsreceiving fluoropyrimidine-based chemotherapy: Evidence from 24studies.” Int. J. Cancer: 123, 2384-2389. Chen, C.-Y., P.-C. Yang, etal. (2011). “Thymidylate synthase and dihydrofolate reductase expressionin non-small cell lung carcinoma: The association with treatmentefficacy of pemetrexed.” Lung Cancer 74(1): 132-138. Lee, S. J., Y. H.Im, et. al. (2010). “Thymidylate synthase and thymidine phosphorylase aspredictive markers of capecitabine monotherapy in patients withanthracycline-and taxane- pretreated metastatic breast cancer.” CancerChemother. Pharmacol. DOI 10.1007/s00280-010- 1545-0. 3 Braun, M. S., M.T. Seymour, et. al. (2008). “Predictive biomarkers of chemotherapyefficacy in colorectal cancer: results from the UK MRC FOCUS trial.” J.Clin. Oncol. 26: 2690-2698. Kostopoulos, I., G. Fountzilas, et. al.(2009). “Topoisomerase I but not thymidylate synthase is associated withimproved outcome in patients with resected colorectal cancer treatedwith irinotecan containing adjuvant chemotherapy.” BMC Cancer. 9: 339.Ataka, M., K. Katano, et. al. (2007). “Topoisomerase I proteinexpression and prognosis of patients with colorectal cancer.” YonagoActa medica. 50: 81-87. 4 Chinot, O. L., M. Barrie, et al. (2007).“Correlation between O6-methylguanine-DNA methyltransferase and survivalin inoperable newly diagnosed glioblastoma patients treated withneoadjuvant temozolomide.” J Clin Oncol 25(12): 1470-5. Kulke, M. H., M.S. Redston, et al. (2008). “06-Methylguanine DNA MethyltransferaseDeficiency and Response to Temozolomide-Based Therapy in Patients withNeuroendocrine Tumors.” Clin Cancer Res 15(1): 338-345. 5 El Sheikh, S.S., H. M. Romanska, et. al. (2008). “Predictive value of PTEN and ARcoexpression of sustained responsiveness to hormonal therapy in prostatecancer--a pilot study.” Neoplasia. 10(9): 949-53. 6 Lewis, J. D., M. J.Edwards, et al. (2010). “Excellent outcomes with adjuvant toremifene ortamoxifen in early stage breast cancer.” Cancer116: 2307-15. Bartlett,J. M. S., D. Rea, et al. (2011). “Estrogen receptor and progesteronereceptor as predictive biomarkers of response to endocrine therapy: aprospectively powered pathology study in the Tamoxifen and ExemestaneAdjuvant Multinational trial.” J Clin Oncol 29 (12): 1531-1538. Dowsett,M., C. Allred, et al. (2008). “Relationship between quantitativeestrogen and progesterone receptor expression and human epidermal growthfactor receptor 2 (HER-2) status with recurrence in the Arimidex,Tamoxifen, Alone or in Combination trial.” J Clin Oncol 26(7): 1059-65.Viale, G., M. M. Regan, et al. (2008). “Chemoendocrine compared withendocrine adjuvant therapies for node-negative breast cancer: predictivevalue of centrally reviewed expression of estrogen and progesteronereceptors--International Breast Cancer Study Group.” J Clin Oncol 26(9):1404-10. Anderson, H., M. Dowsett, et. al. (2011). “Relationship betweenestrogen receptor, progesterone receptor, HER-2 and Ki67 expression andefficacy of aromatase inhibitors in advanced breast cancer. Annals ofOncology. 22: 1770-1776. Coombes, R. C., J. M. Bliss, et al. (2007).“Survival and safety of exemestane versus tamoxifen after 2-3 years'tamoxifen treatment (Intergroup Exemestane Study): a randomizedcontrolled trial.” The Lancet 369: 559-570. Stuart, N. S. 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Any of the biomarker assays herein, including without limitation thoselisted in 7-8 or 12-15, can be performed individually as desired.Additional biomarkers can also be made available for individual testing,e.g., selected from Tables 2 or 6. One of skill will appreciate that anycombination of the individual biomarker assays could be performed. Forexample, a treating physician may choose to order one or more of thefollowing to profile a particular patient's tumor: IHC for at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 or 26 of ALK, AR, cMET, EGFR, ER, ERCC1, H3K36me3,Her2/Neu, MGMT, PBRM1, MLH1, MSH2, MSH6, PD-1, PD-L1, PGP, PMS2, PR,PTEN, RRM1, SPARC, TLE3, TOP2A, TOPO1, TS and TUBB3; ISH (e.g., FISH orCISH) for at least 1, 2, 3, 4, 5, 6, 7 or 8 of 1p19q, ALK, cMET, EGFR,HER2, MDM2, ROS1 and TOP2A; Mutational Analysis of 1, 2, 3 or 4 of BRAF(e.g., Cobas® PCR), IDH2 (e.g., Sanger Sequencing), MGMT-Me (e.g., byPyroSequencing); EGFR (e.g., fragment analysis to detect EGFRvIII); MSIdetection by fragment analysis; and/or Mutational Analysis (e.g., byNext-Generation Sequencing) of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46of ABL1, AKT1, ALK, APC, ATM, BRAF, BRCA1, BRCA2, CDH1, CSF1R, CTNNB1,EGFR, ERBB2 (HER2), ERBB4 (HER4), FBXW7, FGFR1, FGFR2, FLT3, GNA11,GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT (cKIT),KRAS, MET (cMET), MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11,RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL. In some embodiments, aselection of individual tests is made when insufficient tumor sample isavailable for performing all molecular profiling tests in Tables 7-8.

In certain embodiments, ERCC1 is assessed according to the profiles ofthe invention, such as described in any of Tables 7-8. Lack of ERCC1expression, e.g., as determined by IHC, can indicate positive benefitfor platinum compounds (cisplatin, carboplatin, oxaliplatin), andconversely positive expression of ERCC1 can indicate lack of benefit ofthese drugs. The presence of EGFRvIII may be assessed using expressionanalysis at the protein or mRNA level, e.g., by either IHC or PCR,respectively. Expression of EGFRvIII can suggest treatment with EGFRinhibitors. Mutational analysis can be performed for IDH2, e.g., bySanger sequencing, pyrosequencing or by next generation sequencingapproaches. IDH2 mutations suggest the same therapy indications as IDH1mutations, e.g., for decarbazine and temozolomide. In some cases, theanalysis performed for each biomarker can depend on the lineage asdesired. For example, EGFR IHC results may be assessed using H-SCORE forNSCLC but not other lineages.

Additional biomarkers that may be assessed according to the molecularprofiling of the invention include BAP1 (BRCA1 Associated Protein-1(Ubiquitin Carboxy-Terminal Hydrolase)), SETD2 (SET Domain Containing2). In some embodiments of the invention, their expression is assessedat the protein and/or mRNA level. For example, IHC can be used to assessthe protein expression of one or more of these biomarkers. PBRM1 andH3K36me3 may be assessed in kidney cancer, e.g., at the protein levelsuch as by IHC. Molecular profiling of the invention can include atleast one of TOP2A by CISH, Chromosome 17 by CISH, PBRM1 (PB1/BAF180) byIHC, BAP1 by IHC, SETD2 (ANTI-HISTONE H3) by IHC, MDM2 by CISH,Chromosome 12 by CISH, ALK by IHC, CTLA4 by IHC, CD3 by IHC, NY-ESO-1 byIHC, MAGE-A by IHC, TP by IHC, and EGFR by CISH.

Nucleic Acid Mutational Analysis

Nucleic acid analysis may be performed to assess various aspects of agene. For example, nucleic acid analysis can include, but is not limitedto, mutational analysis, fusion analysis, variant analysis, splicevariants, SNP analysis and gene copy number/amplification. Such analysiscan be performed using any number of techniques described herein orknown in the art, including without limitation sequencing (e.g., Sanger,Next Generation, pyrosequencing), PCR, variants of PCR such as RT-PCR,fragment analysis, and the like. NGS techniques may be used to detectmutations, fusions, variants and copy number of multiple genes in asingle assay. Table 6 describes a number of biomarkers including genesbearing mutations that have been identified in various cancer lineages.Unless otherwise stated herein, a “mutation” are used herein maycomprise any change in a gene as compared to its wild type, includingwithout limitation a mutation, polymorphism, deletion, insertion, indels(i.e., insertions or deletions), substitution, translocation, fusion,break, duplication, amplification, repeat, or copy number variation. Inan aspect, the invention provides a molecular profile comprisingmutational analysis of one or more genes in Table 8. In one embodiment,the genes are assessed using Next Generation sequencing methods, e.g.,using a TruSeq/MiSeq/HiSeq/NexSeq system offered by Illumina Corporationor an Ion Torrent system from Life Technologies.

The MI molecular profiles of the invention may comprise mutationalanalysis of additional genes as desired. Exemplary genes are listed inTables 12-15. As desired, different analyses may be performed fordifferent sets of genes. For example, Table 12 lists various genes thatmay be assessed for point mutations and indels, Table 13 lists variousgenes that may be assessed for point mutations, indels and copy numbervariations, Table 14 lists various genes that may be assessed for genefusions, and Table 15 lists genes that can be assessed for transcriptvariants. Gene fusion and transcript analysis may be performed byanalysis of RNA transcripts as desired.

TABLE 12 Point Mutations and Indels ABI1 ABL1 ACKR3 AKT1 AMER1 (FAM123B)AR ARAF ATP2B3 ATRX BCL11B BCL2 BCL2L2 BCOR BCORL1 BRD3 BRD4 BTG1 BTKC15orf65 CBLC CD79B CDH1 CDK12 CDKN2B CDKN2C CEBPA CHCHD7 CNOT3 COL1A1COX6C CRLF2 DDB2 DDIT3 DNM2 DNMT3A EIF4A2 ELF4 ELN ERCC1 ETV4 FAM46CFANCF FEV FOXL2 FOXO3 FOXO4 FSTL3 GATA1 GATA2 GNA11 GPC3 HEY1 HIST1H3BHIST1H4I HLF HMGN2P46 HNF1A HOXA11 HOXA13 HOXA9 HOXC11 HOXC13 HOXD11HOXD13 HRAS IKBKE INHBA IRS2 JUN KAT6A (MYST3) KAT6B KCNJ5 KDM5C KDM6AKDSR KLF4 KLK2 LASP1 LMO1 LMO2 MAFB MAX MECOM MED12 MKL1 MLLT11 MN1 MPLMSN MTCP1 MUC1 MUTYH MYCL (MYCL1) NBN NDRG1 NKX2-1 NONO NOTCH1 NRASNUMA1 NUTM2B OLIG2 OMD P2RY8 PAFAH1B2 PAK3 PATZ1 PAX8 PDE4DIP PHF6PHOX2B PIK3CG PLAG1 PMS1 POU5F1 PPP2R1A PRF1 PRKDC RAD21 RECQL4 RHOHRNF213 RPL10 SEPT5 SEPT6 SFPQ SLC45A3 SMARCA4 SOCS1 SOX2 SPOP SRC SSX1STAG2 TAL1 TAL2 TBL1XR1 TCEA1 TCL1A TERT TFE3 TFPT THRAP3 TLX3 TMPRSS2UBR5 VHL WAS ZBTB16 ZRSR2

TABLE 13 Point Mutations, Indels and Copy Number Variations ABL2 ACSL3ACSL6 AFF1 AFF3 AFF4 AKAP9 AKT2 AKT3 ALDH2 ALK APC ARFRP1 ARHGAP26ARHGEF12 ARID1A ARID2 ARNT ASPSCR1 ASXL1 ATF1 ATIC ATM ATP1A1 ATR AURKAAURKB AXIN1 AXL BAP1 BARD1 BCL10 BCL11A BCL2L11 BCL3 BCL6 BCL7A BCL9 BCRBIRC3 BLM BMPR1A BRAF BRCA1 BRCA2 BRIP1 BUB1B C11orf30 (EMSY) C2orf44CACNA1D CALR CAMTA1 CANT1 CARD11 CARS CASC5 CASP8 CBFA2T3 CBFB CBL CBLBCCDC6 CCNB1IP1 CCND1 CCND2 CCND3 CCNE1 CD274 (PDL1) CD74 CD79A CDC73CDH11 CDK4 CDK6 CDK8 CDKN1B CDKN2A CDX2 CHEK1 CHEK2 CHIC2 CHN1 CIC CITTACLP1 CLTC CLTCL1 CNBP CNTRL COPB1 CREB1 CREB3L1 CREB3L2 CREBBP CRKLCRTC1 CRTC3 CSF1R CSF3R CTCF CTLA4 CTNNA1 CTNNB1 CYLD CYP2D6 DAXX DDR2DDX10 DDX5 DDX6 DEK DICER1 DOT1L EBF1 ECT2L EGFR ELK4 ELL EML4 EP300EPHA3 EPHA5 EPHB1 EPS15 ERBB2 (HER2) ERBB3 (HER3) ERBB4 (HER4) ERC1ERCC2 ERCC3 ERCC4 ERCC5 ERG ESR1 ETV1 ETV5 ETV6 EWSR1 EXT1 EXT2 EZH2 EZRFANCA FANCC FANCD2 FANCE FANCG FANCL FAS FBXO11 FBXW7 FCRL4 FGF10 FGF14FGF19 FGF23 FGF3 FGF4 FGF6 FGFR1 FGFR1OP FGFR2 FGFR3 FGFR4 FH FHITFIP1L1 FLCN FLI1 FLT1 FLT3 FLT4 FNBP1 FOXA1 FOXO1 FOXP1 FUBP1 FUS GAS7GATA3 GID4 (C17orf39) GMPS GNA13 GNAQ GNAS GOLGA5 GOPC GPHN GPR124GRIN2A GSK3B H3F3A H3F3B HERPUD1 HGF HIP1 HMGA1 HMGA2 HNRNPA2B1 HOOK3HSP90AA1 HSP90AB1 IDH1 IDH2 IGF1R IKZF1 IL2 IL21R IL6ST IL7R IRF4 ITKJAK1 JAK2 JAK3 JAZF1 KDM5A KDR (VEGFR2) KEAP1 KIAA1549 KIF5B KIT KLHL6KMT2A (MLL) KMT2C (MLL3) KMT2D (MLL2) KRAS KTN1 LCK LCP1 LGR5 LHFP LIFRLPP LRIG3 LRP1B LYL1 MAF MALT1 MAML2 MAP2K1 MAP2K2 MAP2K4 MAP3K1 MCL1MDM2 MDM4 MDS2 MEF2B MEN1 MET (cMET) MITF MLF1 MLH1 MLLT1 MLLT10 MLLT3MLLT4 MLLT6 MNX1 MRE11A MSH2 MSH6 MSI2 MTOR MYB MYC MYCN MYD88 MYH11MYH9 NACA NCKIPSD NCOA1 NCOA2 NCOA4 NF1 NF2 NFE2L2 NFIB NFKB2 NFKBIA NINNOTCH2 NPM1 NR4A3 NSD1 NT5C2 NTRK1 NTRK2 NTRK3 NUP214 NUP93 NUP98 NUTM1PALB2 PAX3 PAX5 PAX7 PBRM1 PBX1 PCM1 PCSK7 PDCD1 (PD1) PDCD1LG2 (PDL2)PDGFB PDGFRA PDGFRB PDK1 PER1 PICALM PIK3CA PIK3R1 PIK3R2 PIM1 PML PMS2POLE POT1 POU2AF1 PPARG PRCC PRDM1 PRDM16 PRKAR1A PRRX1 PSIP1 PTCH1 PTENPTPN11 PTPRC RABEP1 RAC1 RAD50 RADS1 RAD51B RAF1 RALGDS RANBP17 RAP1GDS1RARA RB1 RBM15 REL RET RICTOR RMI2 RNF43 ROS1 RPL22 RPL5 RPN1 RPTORRUNX1 RUNX1T1 SBDS SDC4 SDHAF2 SDHB SDHC SDHD SEPT9 SET SETBP1 SETD2SF3B1 SH2B3 SH3GL1 SLC34A2 SMAD2 SMAD4 SMARCB1 SMARCE1 SMO SNX29 SOX10SPECC1 SPEN SRGAP3 SRSF2 SRSF3 SS18 5518L1 STAT3 STAT4 STAT5B STIL STK11SUFU SUZ12 SYK TAF15 TCF12 TCF3 TCF7L2 TET1 TET2 TFEB TFG TFRC TGFBR2TLX1 TNFAIP3 TNFRSF14 TNFRSF17 TOP1 TP53 TPM3 TPM4 TPR TRAF7 TRIM26TRIM27 TRIM33 TRIP11 TRRAP TSC1 TSC2 TSHR TTL U2AF1 USP6 VEGFA VEGFBVTI1A WHSC1 WHSC1L1 WIF1 WISP3 WRN WT1 WWTR1 XPA XPC XPO1 YWHAE ZMYM2ZNF217 ZNF331 ZNF384 ZNF521 ZNF703

TABLE 14 Gene Fusions ALK BRAF NTRK1 NTRK2 NTRK3 RET ROS1 RSPO3

TABLE 15 Variant Transcripts EGFR vIII MET Exon 14 Skipping

In an aspect, the invention provides a molecular profile for a cancerwhich comprises mutational analysis of a panel of genes. In someembodiments, the panel of genes is selected from Table 8 as describedherein. For example, the molecular profile may comprise mutationalanalysis of at least one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or46, of ABL1, AKT1, ALK, APC, ATM, BRAF, BRCA1, BRCA2, CDH1, CSF1R,CTNNB1, EGFR, ERBB2 (HER2), ERBB4 (HER4), FBXW7, FGFR1, FGFR2, FLT3,GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT(cKIT), KRAS, MET (cMET), MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN,PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11, TP53, and VHL. The statusof the genes can be linked to drug efficacy (e.g., predicted benefit orlack of benefit) or clinical trial enrollment as desired. See, e.g.,Table 9.

In other embodiments, the panel of genes assessed as part of the MImolecular profiling is expanded to include additional biomarkers. Such amolecular profile may be referred to as an “MI Profile X” profile. In anembodiment, the additional biomarkers assessed by mutational analysisinclude at least one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550 or allgenes listed in Tables 12-15. The molecular profile may compriseanalysis of at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, orall of ABI1, ABL1, ACKR3, AKT1, AMER1 (FAM123B), AR, ARAF, ATP2B3, ATRX,BCL11B, BCL2, BCL2L2, BCOR, BCORL1, BRD3, BRD4, BTG1, BTK, C15orf65,CBLC, CD79B, CDH1, CDK12, CDKN2B, CDKN2C, CEBPA, CHCHD7, CNOT3, COL1A1,COX6C, CRLF2, DDB2, DDIT3, DNM2, DNMT3A, EIF4A2, ELF4, ELN, ERCC1, ETV4,FAM46C, FANCF, FEV, FOXL2, FOXO3, FOXO4, FSTL3, GATA1, GATA2, GNA11,GPC3, HEY1, HIST1H3B, HIST1H4I, HLF, HMGN2P46, HNF1A, HOXA11, HOXA13,HOXA9, HOXC11, HOXC13, HOXD11, HOXD13, HRAS, IKBKE, INHBA, IRS2, JUN,KAT6A (MYST3), KAT6B, KCNJ5, KDM5C, KDM6A, KDSR, KLF4, KLK2, LASP1,LMO1, LMO2, MAFB, MAX, MECOM, MED12, MKL1, MLLT11, MN1, MPL, MSN, MTCP1,MUC1, MUTYH, MYCL (MYCL1), NBN, NDRG1, NKX2-1, NONO, NOTCH1, NRAS,NUMA1, NUTM2B, OLIG2, OMD, P2RY8, PAFAH1B2, PAK3, PATZ1, PAX8, PDE4DIP,PHF6, PHOX2B, PIK3CG, PLAG1, PMS1, POU5F1, PPP2R1A, PRF1, PRKDC, RAD21,RECQL4, RHOH, RNF213, RPL10, SEPT5, SEPT6, SFPQ, SLC45A3, SMARCA4,SOCS1, SOX2, SPOP, SRC, SSX1, STAG2, TAL1, TAL2, TBL1XR1, TCEA1, TCL1A,TERT, TFE3, TFPT, THRAP3, TLX3, TMPRSS2, UBR5, VHL, WAS, ZBTB16 andZRSR2. Such genes can be assessed, e.g., for point mutations and indels,or other characteristics as desired. The molecular profile may compriseanalysis of at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 350, 400 or all, of ABL2, ACSL3,ACSL6, AFF1, AFF3, AFF4, AKAP9, AKT2, AKT3, ALDH2, ALK, APC, ARFRP1,ARHGAP26, ARHGEF12, ARID1A, ARID2, ARNT, ASPSCR1, ASXL1, ATF1, ATIC,ATM, ATP1A1, ATR, AURKA, AURKB, AXIN1, AXL, BAP1, BARD1, BCL10, BCL11A,BCL2L11, BCL3, BCL6, BCL7A, BCL9, BCR, BIRC3, BLM, BMPR1A, BRAF, BRCA1,BRCA2, BRIP1, BUB1B, C11orf30 (EMSY), C2orf44, CACNA1D, CALR, CAMTA1,CANT1, CARD11, CARS, CASC5, CASP8, CBFA2T3, CBFB, CBL, CBLB, CCDC6,CCNB1IP1, CCND1, CCND2, CCND3, CCNE1, CD274 (PDL1), CD74, CD79A, CDC73,CDH11, CDK4, CDK6, CDK8, CDKN1B, CDKN2A, CDX2, CHEK1, CHEK2, CHIC2,CHN1, CIC, CIITA, CLP1, CLTC, CLTCL1, CNBP, CNTRL, COPB1, CREB1,CREB3L1, CREB3L2, CREBBP, CRKL, CRTC1, CRTC3, CSF1R, CSF3R, CTCF, CTLA4,CTNNA1, CTNNB1, CYLD, CYP2D6, DAXX, DDR2, DDX10, DDX5, DDX6, DEK,DICER1, DOT1L, EBF1, ECT2L, EGFR, ELK4, ELL, EML4, EP300, EPHA3, EPHA5,EPHB1, EPS15, ERBB2 (HER2), ERBB3 (HER3), ERBB4 (HER4), ERC1, ERCC2,ERCC3, ERCC4, ERCC5, ERG, ESR1, ETV1, ETV5, ETV6, EWSR1, EXT1, EXT2,EZH2, EZR, FANCA, FANCC, FANCD2, FANCE, FANCG, FANCL, FAS, FBXO11,FBXW7, FCRL4, FGF10, FGF14, FGF19, FGF23, FGF3, FGF4, FGF6, FGFR1,FGFR1OP, FGFR2, FGFR3, FGFR4, FH, FHIT, FIP1L1, FLCN, FLI1, FLT1, FLT3,FLT4, FNBP1, FOXA1, FOXO1, FOXP1, FUBP1, FUS, GAS7, GATA3, GID4(C17orf39), GMPS, GNA13, GNAQ, GNAS, GOLGA5, GOPC, GPHN, GPR124, GRIN2A,GSK3B, H3F3A, H3F3B, HERPUD1, HGF, HIP1, HMGA1, HMGA2, HNRNPA2B1, HOOK3,HSP90AA1, HSP90AB1, IDH1, IDH2, IGF1R, IKZF1, IL2, IL21R, IL6ST, IL7R,IRF4, ITK, JAK1, JAK2, JAK3, JAZF1, KDM5A, KDR (VEGFR2), KEAP1,KIAA1549, KIF5B, KIT, KLHL6, KMT2A (MLL), KMT2C (MLL3), KMT2D (MLL2),KRAS, KTN1, LCK, LCP1, LGR5, LHFP, LIFR, LPP, LRIG3, LRP1B, LYL1, MAF,MALT1, MAML2, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MCL1, MDM2, MDM4, MDS2,MEF2B, MEN1, MET (cMET), MITF, MLF1, MLH1, MLLT1, MLLT10, MLLT3, MLLT4,MLLT6, MNX1, MRE11A, MSH2, MSH6, MSI2, MTOR, MYB, MYC, MYCN, MYD88,MYH11, MYH9, NACA, NCKIPSD, NCOA1, NCOA2, NCOA4, NF1, NF2, NFE2L2, NFIB,NFKB2, NFKBIA, NIN, NOTCH2, NPM1, NR4A3, NSD1, NT5C2, NTRK1, NTRK2,NTRK3, NUP214, NUP93, NUP98, NUTM1, PALB2, PAX3, PAX5, PAX7, PBRM1,PBX1, PCM1, PCSK7, PDCD1 (PD1), PDCD1LG2 (PDL2), PDGFB, PDGFRA, PDGFRB,PDK1, PER1, PICALM, PIK3CA, PIK3R1, PIK3R2, PIM1, PML, PMS2, POLE, POT1,POU2AF1, PPARG, PRCC, PRDM1, PRDM16, PRKAR1A, PRRX1, PSIP1, PTCH1, PTEN,PTPN11, PTPRC, RABEP1, RAC1, RAD50, RAD51, RAD51B, RAF1, RALGDS,RANBP17, RAP1GDS1, RARA, RB1, RBM15, REL, RET, RICTOR, RMI2, RNF43,ROS1, RPL22, RPL5, RPN1, RPTOR, RUNX1, RUNX1T1, SBDS, SDC4, SDHAF2,SDHB, SDHC, SDHD, SEPT9, SET, SETBP1, SETD2, SF3B1, SH2B3, SH3GL1,SLC34A2, SMAD2, SMAD4, SMARCB1, SMARCE1, SMO, SNX29, SOX10, SPECC1,SPEN, SRGAP3, SRSF2, SRSF3, SS18, SS18L1, STAT3, STAT4, STATSB, STIL,STK11, SUFU, SUZ12, SYK, TAF15, TCF12, TCF3, TCF7L2, TET1, TET2, TFEB,TFG, TFRC, TGFBR2, TLX1, TNFAIP3, TNFRSF14, TNFRSF17, TOP1, TP53, TPM3,TPM4, TPR, TRAF7, TRIM26, TRIM27, TRIM33, TRIP11, TRRAP, TSC1, TSC2,TSHR, TTL, U2AF1, USP6, VEGFA, VEGFB, VTI1A, WHSC1, WHSC1L1, WIF1,WISP3, WRN, WT1, WWTR1, XPA, XPC, XPO1, YWHAE, ZMYM2, ZNF217, ZNF331,ZNF384, ZNF521 and ZNF703. Such genes can be assessed, e.g., for pointmutations, indels and copy number, or other characteristics as desired.The molecular profile may comprise analysis of at least one, e.g., 1, 2,3, 4, 5, 6, 7 or 8 of ALK, BRAF, NTRK1, NTRK2, NTRK3, RET, ROS1 andRSPO3. Such genes can be assessed for gene fusions or othercharacteristics as desired. The molecular profile may comprise analysisof EGFR vIII and/or MET Exon 14 Skipping. Such analysis may includeidentification of variant transcripts. In some embodiments, all geneslisted in Tables 12-15 are analyzed as indicated in the table headers.NGS sequencing may be used to perform such analysis in a high throughputmanner. Any useful combinations such as those listed in this paragraphmay be assessed by mutational analysis.

In an embodiment, the plurality of genes and/or gene products comprisesmutational analysis of at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, of ABL1, AKT1,ALK, APC, AR, ARAF, ATM, BAP1, BRAF, BRCA1, BRCA2, CDK4, CDKN2A, CHEK1,CHEK2, CSF1R, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, FGFR1, FGFR2, FGFR3,FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, JAK2, KDR, KIT, KRAS, MAP2K1(MEK1), MAP2K2 (MEK2), MET, MLH1, MPL, NF1, NOTCH1, NRAS, NTRK1, PDGFRA,PDGFRB, PIK3CA, PTCH1, PTEN, RAF1, RET, ROS1, SMO, SRC, TP53, VHL, WT1.The genes assessed by mutational analysis may further comprise at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400,450, 500, or all genes, selected from the group consisting of ABI1,ABL2, ACSL3, ACSL6, AFF1, AFF3, AFF4, AKAP9, AKT2, AKT3, ALDH2, AMER1,AR, ARFRP1, ARHGAP26, ARHGEF12, ARID1A, ARID2, ARNT, ASPSCR1, ASXL1,ATF1, ATIC, ATP1A1, ATP2B3, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, BARD1,BCL10, BCL11A, BCL11B, BCL2, BCL2L11, BCL2L2, BCL3, BCL6, BCL7A, BCL9,BCOR, BCORL1, BCR, BIRC3, BLM, BMPR1A, BRD3, BRD4, BRIP1, BTG1, BTK,BUB1B, C11orf30, C15orf21, C15orf55, C15orf65, C16orf75, C2orf44,CACNA1D, CALR, CAMTA1, CANT1, CARD11, CARS, CASC5, CASP8, CBFA2T3, CBFB,CBL, CBLB, CBLC, CCDC6, CCNB1IP1, CCND1, CCND2, CCND3, CCNE1, CD274,CD74, CD79A, CD79B, CDC73, CDH11, CDK12, CDK4, CDK6, CDK8, CDKN1B,CDKN2A, CDKN2B, CDKN2C, CDX2, CEBPA, CHCHD7, CHIC2, CHN1, CIC, CIITA,CLP1, CLTC, CLTCL1, CNBP, CNOT3, CNTRL, COL1A1, COPB1, COX6C, CREB1,CREB3L1, CREB3L2, CREBBP, CRKL, CRLF2, CRTC1, CRTC3, CSF3R, CTCF, CTLA4,CTNNA1, CXCR7, CYLD, CYP2D6, DAXX, DDB2, DDIT3, DDX10, DDX5, DDX6, DEK,DICER1, DNM2, DNMT3A, DOT1L, DUX4, EBF1, ECT2L, EIF4A2, ELF4, ELK4, ELL,ELN, EML4, EP300, EPHA3, EPHA5, EPHB1, EPS15, ERC1, ERCC1, ERCC2, ERCC3,ERCC4, ERCC5, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EWSR1, EXT1, EXT2,EZH2, EZR, FAM123B, FAM22A, FAM22B, FAM46C, FANCA, FANCC, FANCD2, FANCE,FANCF, FANCG, FANCL, FAS, FBXO11, FCGR2B, FCRL4, FEV, FGF10, FGF14,FGF19, FGF23, FGF3, FGF4, FGF6, FGFR1OP, FGFR3, FGFR4, FH, FHIT, FIP1L1,FLCN, FLI1, FLT1, FLT4, FNBP1, FOXA1, FOXL2, FOXO1, FOXO3, FOXO4, FOXP1,FSTL3, FUBP1, FUS, GAS7, GATA1, GATA2, GATA3, GID4, GMPS, GNA13, GOLGA5,GOPC, GPC3, GPHN, GPR124, GRIN2A, GSK3B, H3F3A, H3F3B, HERPUD1, HEY1,HGF, HIP1, HIST1H3B, HIST1H4I, HLF, HMGA1, HMGA2, HNRNPA2B1, HOOK3,HOXA11, HOXA13, HOXA9, HOXC11, HOXC13, HOXD11, HOXD13, HSP90AA1,HSP90AB1, IGF1R, IKBKE, IKZF1, IL2, IL21R, IL6ST, IL7R, INHBA, IRF4,IRS2, ITK, JAK1, JAZF1, JUN, KAT6A, KCNJ5, KDM5A, KDM5C, KDM6A, KDSR,KEAP1, KIAA1549, KIF5B, KLF4, KLHL6, KLK2, KTN1, LASP1, LCK, LCP1, LGR5,LHFP, LIFR, LMO1, LMO2, LPP, LRIG3, LRP1B, LYL1, MAF, MAFB, MALT1,MAML2, MAP2K1 (MEK1), MAP2K2 (MEK2), MAP2K4, MAP3K1, MAX, MCL1, MDM2,MDM4, MDS2, MECOM, MED12, MEF2B, MEN1, MITF, MKL1, MLF1, MLL, MLL2,MLL3, MLLT1, MLLT10, MLLT11, MLLT3, MLLT4, MLLT6, MN1, MNX1, MRE11A,MSH2, MSH6, MSI2, MSN, MTCP1, MTOR, MUC1, MUTYH, MYB, MYC, MYCL1, MYCN,MYD88, MYH11, MYH9, MYST4, NACA, NBN, NCKIPSD, NCOA1, NCOA2, NCOA4,NDRG1, NF2, NFE2L2, NFIB, NFKB2, NFKBIA, NIN, NKX2-1, NONO, NOTCH2,NR4A3, NSD1, NT5C2, NTRK2, NTRK3, NUMA1, NUP214, NUP93, NUP98, OLIG2,OMD, P2RY8, PAFAH1B2, PAK3, PALB2, PATZ1, PAX3, PAX5, PAX7, PAX8, PBRM1,PBX1, PCM1, PCSK7, PDCD1, PDCD1LG2, PDE4DIP, PDGFB, PDGFRB, PDK1, PER1,PHF6, PHOX2B, PICALM, PIK3CG, PIK3R1, PIK3R2, PIM1, PLAG1, PML, PMS1,PMS2, POLE, POT1, POU2AF1, POU5F1, PPARG, PPP2R1A, PRCC, PRDM1, PRDM16,PRF1, PRKAR1A, PRKDC, PRRX1, PSIP1, PTCH1, PTPRC, RABEP1, RAC1, RAD21,RAD50, RAD51, RADSIL1, RALGDS, RANBP17, RAP1GDS1, RARA, RBM15, RECQL4,REL, RHOH, RICTOR, RNF213, RNF43, RPL10, RPL22, RPL5, RPN1, RPTOR,RUNDC2A, RUNX1, RUNx1T1, SBDS, SDC4, SDHAF2, SDHB, SDHC, SDHD, SEPT5,SEPT6, SEPT9, SET, SETBP1, SETD2, SF3B1, SFPQ, SFRS3, SH2B3, SH3GL1,SLC34A2, SLC45A3, SMAD2, SMARCA4, SMARCE1, SOCS1, SOX10, SOX2, SPECC1,SPEN, SPOP, SRC, SRGAP3, SRSF2, SS18, SS18L1, SSX1, SSX2, SSX4, STAG2,STAT3, STAT4, STAT5B, STIL, SUFU, SUZ12, SYK, TAF15, TAL1, TAL2,TBL1XR1, TCEA1, TCF12, TCF3, TCF7L2, TCL1A, TERT, TET1, TET2, TFE3,TFEB, TFG, TFPT, TFRC, TGFBR2, THRAP3, TLX1, TLX3, TMPRSS2, TNFAIP3,TNFRSF14, TNFRSF17, TOP1, TPM3, TPM4, TPR, TRAF7, TRIM26, TRIM27,TRIM33, TRIP11, TRRAP, TSC1, TSC2, TSHR, TTL, U2AF1, UBR5, USP6, VEGFA,VEGFB, VTI1A, WAS, WHSC1, WHSC1L1, WIF1, WISP3, WRN, WWTR1, XPA, XPC,XPO1, YWHAE, ZBTB16, ZMYM2, ZNF217, ZNF331, ZNF384, ZNF521, ZNF703 andZRSR2. Any useful combinations such as those listed in this paragraphmay be assessed by mutational analysis.

The genes assessed by mutational analysis may comprise at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, orall genes, selected from the group consisting of ABI1, ABL1, ABL2,ACKR3, ACSL3, ACSL6, AFF1, AFF3, AFF4, AKAP9, AKT1, AKT2, AKT3, ALDH2,ALK, AMER1 (FAM123B), APC, AR, ARAF, ARFRP1, ARHGAP26, ARHGEF12, ARID1A,ARID2, ARNT, ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATP1A1, ATP2B3, ATR, ATRX,AURKA, AURKB, AXIN1, AXL, BAP1, BARD1, BCL10, BCL11A, BCL11B, BCL2,BCL2L11, BCL2L2, BCL3, BCL6, BCL7A, BCL9, BCOR, BCORL1, BCR, BIRC3, BLM,BMPR1A, BRAF, BRCA1, BRCA2, BRD3, BRD4, BRIP1, BTG1, BTK, BUB1B,C11orf30 (EMSY), C15orf65, C2orf44, CACNA1D, CALK, CAMTA1, CANT1,CARD11, CARS, CASC5, CASP8, CBFA2T3, CBFB, CBL, CBLB, CBLC, CCDC6,CCNB1IP1, CCND1, CCND2, CCND3, CCNE1, CD274 (PDL1), CD74, CD79A, CD79B,CDC73, CDH1, CDH11, CDK12, CDK4, CDK6, CDK8, CDKN1B, CDKN2A, CDKN2B,CDKN2C, CDX2, CEBPA, CHCHD7, CHEK1, CHEK2, CHIC2, CHN1, CIC, CIITA,CLP1, CLTC, CLTCL1, CNBP, CNOT3, CNTRL, COL1A1, COPB1, COX6C, CREB1,CREB3L1, CREB3L2, CREBBP, CRKL, CRLF2, CRTC1, CRTC3, CSF1R, CSF3R, CTCF,CTLA4, CTNNA1, CTNNB1, CYLD, CYP2D6, DAXX, DDB2, DDIT3, DDR2, DDX10,DDX5, DDX6, DEK, DICER1, DNM2, DNMT3A, DOT1L, EBF1, ECT2L, EGFR, EIF4A2,ELF4, ELK4, ELL, ELN, EML4, EP300, EPHA3, EPHA5, EPHB1, EPS15, ERBB2(HER2), ERBB3 (HER3), ERBB4 (HER4), ERC1, ERCC1, ERCC2, ERCC3, ERCC4,ERCC5, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EWSR1, EXT1, EXT2, EZH2, EZR,FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, FAS, FBXO11,FBXW7, FCRL4, FEV, FGF10, FGF14, FGF19, FGF23, FGF3, FGF4, FGF6, FGFR1,FGFR1OP, FGFR2, FGFR3, FGFR4, FH, FHIT, FIP1L1, FLCN, FLI1, FLT1, FLT3,FLT4, FNBP1, FOXA1, FOXL2, FOXO1, FOXO3, FOXO4, FOXP1, FSTL3, FUBP1,FUS, GAS7, GATA1, GATA2, GATA3, GID4 (C17orf39), GMPS, GNA11, GNA13,GNAQ, GNAS, GOLGA5, GOPC, GPC3, GPHN, GPR124, GRIN2A, GSK3B, H3F3A,H3F3B, HERPUD1, HEY1, HGF, HIP1, HIST1H3B, HIST1H4I, HLF, HMGA1, HMGA2,HMGN2P46, HNF1A, HNRNPA2B1, HOOK3, HOXA11, HOXA13, HOXA9, HOXC11,HOXC13, HOXD11, HOXD13, HRAS, HSP90AA1, HSP90AB1, IDH1, IDH2, IGF1R,IKBKE, IKZF1, IL2, IL21R, IL6ST, IL7R, INHBA, IRF4, IRS2, ITK, JAK1,JAK2, JAK3, JAZF1, JUN, KAT6A (MYST3), KAT6B, KCNJ5, KDM5A, KDM5C,KDM6A, KDR, KDSR, KEAP1, KIAA1549, KIF5B, KIT, KLF4, KLHL6, KLK2, KMT2A(MLL), KMT2C (MLL3), KMT2D (MLL2), KRAS, KTN1, LASP1, LCK, LCP1, LGR5,LHFP, LIFR, LMO1, LMO2, LPP, LRIG3, LRP1B, LYL1, MAF, MAFB, MALT1,MAML2, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAX, MCL1, MDM2, MDM4, MDS2,MECOM, MED12, MEF2B, MEN1, MET, MITF, MKL1, MLF1, MLH1, MLLT1, MLLT10,MLLT11, MLLT3, MLLT4, MLLT6, MN1, MNX1, MPL, MRE11A, MSH2, MSH6, MSI2,MSN, MTCP1, MTOR, MUC1, MUTYH, MYB, MYC, MYCL (MYCL1), MYCN, MYD88,MYH11, MYH9, NACA, NBN, NCKIPSD, NCOA1, NCOA2, NCOA4, NDRG1, NF1, NF2,NFE2L2, NFIB, NFKB2, NFKBIA, NIN, NKX2-1, NONO, NOTCH1, NOTCH2, NPM1,NR4A3, NRAS, NSD1, NT5C2, NTRK1, NTRK2, NTRK3, NUMA1, NUP214, NUP93,NUP98, NUTM1, NUTM2B, OLIG2, OMD, P2RY8, PAFAH1B2, PAK3, PALB2, PATZ1,PAX3, PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1, PCSK7, PDCD1 (PD1), PDCD1LG2(PDL2), PDE4DIP, PDGFB, PDGFRA, PDGFRB, PDK1, PER1, PHF6, PHOX2B,PICALM, PIK3CA, PIK3CG, PIK3R1, PIK3R2, PIM1, PLAG1, PML, PMS1, PMS2,POLE, POT1, POU2AF1, POU5F1, PPARG, PPP2R1A, PRCC, PRDM1, PRDM16, PRF1,PRKAR1A, PRKDC, PRRX1, PSIP1, PTCH1, PTEN, PTPN11, PTPRC, RABEP1, RAC1,RAD21, RAD50, RAD51, RAD51B, RAF1, RALGDS, RANBP17, RAP1GDS1, RARA, RB1,RBM15, RECQL4, REL, RET, RHOH, RICTOR, RMI2, RNF213, RNF43, ROS1, RPL10,RPL22, RPL5, RPN1, RPTOR, RSPO3, RUNX1, RUNx1T1, SBDS, SDC4, SDHAF2,SDHB, SDHC, SDHD, SEPT5, SEPT6, SEPT9, SET, SETBP1, SETD2, SF3B1, SFPQ,SH2B3, SH3GL1, SLC34A2, SLC45A3, SMAD2, SMAD4, SMARCA4, SMARCB1,SMARCE1, SMO, SNX29, SOCS1, SOX10, SOX2, SPECC1, SPEN, SPOP, SRC,SRGAP3, SRSF2, SRSF3, SS18, SS18L1, SSX1, STAG2, STAT3, STAT4, STATSB,STIL, STK11, SUFU, SUZ12, SYK, TAF15, TAL1, TAL2, TBL1XR1, TCEA1, TCF12,TCF3, TCF7L2, TCL1A, TERT, TET1, TET2, TFE3, TFEB, TFG, TFPT, TFRC,TGFBR2, THRAP3, TLX1, TLX3, TMPRSS2, TNFAIP3, TNFRSF14, TNFRSF17, TOP1,TP53, TPM3, TPM4, TPR, TRAF7, TRIM26, TRIM27, TRIM33, TRIP11, TRRAP,TSC1, TSC2, TSHR, TTL, U2AF1, UBR5, USP6, VEGFA, VEGFB, VHL, VTI1A, WAS,WHSC1, WHSC1L1, WIF1, WISP3, WRN, WT1, WWTR1, XPA, XPC, XPO1, YWHAE,ZBTB16, ZMYM2, ZNF217, ZNF331, ZNF384, ZNF521, ZNF703 and ZRSR2. Anyuseful combinations such as those listed in this paragraph may beassessed by mutational analysis.

The genes assessed by mutational analysis may comprise at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, or all genes, selected from the group consisting ofABCB1, ABCG2, ABI1, ABL1, ABL2, ACKR3, ACSL3, ACSL6, ACVR1B, ACVR2A,AFF1, AFF3, AFF4, AKAP9, AKT1, AKT2, AKT3, ALDH1A1, ALDH2, ALK, AMER1,ANGPT1, ANGPT2, ANKRD23, APC, AR, ARAF, AREG, ARFRP1, ARHGAP26,ARHGEF12, ARID1A, ARID1B, ARID2, ARNT, ASPSCR1, ASXL1, ATF1, ATIC, ATM,ATP1A1, ATP2B3, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, BAP1, BARD1, BBC3,BCL10, BCL11A, BCL11B, BCL2, BCL2L1, BCL2L11, BCL2L2, BCL3, BCL6, BCL7A,BCL9, BCOR, BCORL1, BCR, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD3,BRD4, BRINP3, BRIP1, BTG1, BTG2, BTK, BUB1B, C11orf30, C15orf65,C2orf44, CA6, CACNA1D, CALK, CAMTA1, CANT1, CARD11, CARS, CASC5, CASP8,CBFA2T3, CBFB, CBL, CBLB, CBLC, CCDC6, CCNBlIP1, CCND1, CCND2, CCND3,CCNE1, CD19, CD22, CD274, CD38, CD4, CD70, CD74, CD79A, CD79B, CD83,CDC73, CDH1, CDH11, CDK12, CDK4, CDK6, CDK7, CDK8, CDK9, CDKN1A, CDKN1B,CDKN2A, CDKN2B, CDKN2C, CDX2, CEBPA, CHCHD7, CHD2, CHD4, CHEK1, CHEK2,CHIC2, CHN1, CHORDC1, CIC, CIITA, CLP1, CLTC, CLTCL1, CNBP, CNOT3,CNTRL, COL1A1, COPB1, COX6C, CRBN, CREB1, CREB3L1, CREB3L2, CREBBP,CRKL, CRLF2, CRTC1, CRTC3, CSF1R, CSF3R, CTCF, CTLA4, CTNNA1, CTNNB1,CUL3, CXCR4, CYLD, CYP17A1, CYP2D6, DAXX, DDB2, DDIT3, DDR1, DDR2,DDX10, DDX3X, DDX5, DDX6, DEK, DICER1, DIS3, DLL4, DNM2, DNMT1, DNMT3A,DOT1L, DPYD, DUSP4, DUSP6, EBF1, ECT2L, EDNRB, EED, EGFR, EIF4A2, ELF4,ELK4, ELL, ELN, EML4, EP300, EPHA3, EPHA5, EPHA7, EPHA8, EPHB1, EPHB2,EPHB4, EPS15, ERBB2, ERBB3, ERBB4, ERC1, ERCC1, ERCC2, ERCC3, ERCC4,ERCC5, EREG, ERG, ERN1, ERRFI1, ESR1, ETV1, ETV4, ETV5, ETV6, EWSR1,EXT1, EXT2, EZH2, EZR, FAF1, FAIM3, FAM46C, FANCA, FANCC, FANCD2, FANCE,FANCF, FANCG, FANCL, FAS, FAT1, FBXO11, FBXW7, FCRL4, FEV, FGF10, FGF14,FGF19, FGF2, FGF23, FGF3, FGF4, FGF6, FGFR1, FGFR1OP, FGFR2, FGFR3,FGFR4, FH, FHIT, FIP1L1, FKBP1A, FLCN, FLI1, FLT1, FLT3, FLT4, FNBP1,FOXA1, FOXL2, FOXO1, FOXO3, FOXO4, FOXP1, FRS2, FSTL3, FUBP1, FUS,GABRA6, GAS7, GATA1, GATA2, GATA3, GATA4, GATA6, GID4, GLI1, GMPS,GNA11, GNA12, GNA13, GNAQ, GNAS, GNRH1, GOLGA5, GOPC, GPC3, GPHN,GPR124, GRIN2A, GRM3, GSK3B, GUCY2C, H3F3A, H3F3B, HCK, HDAC1, HERPUD1,HEY1, HGF, HIP1, HIST1H1E, HIST1H3B, HIST1H4I, HLF, HMGA1, HMGA2,HMGN2P46, HNF1A, HNMT, HNRNPA2B1, HNRNPK, HOOK3, HOXA11, HOXA13, HOXA9,HOXC11, HOXC13, HOXD11, HOXD13, HRAS, HSD3B1, HSP90AA1, HSP90AB1, IAPP,ID3, IDH1, IDH2, IGF1R, IGF2, IKBKE, IKZF1, IL2, IL21R, IL3RA, IL6,IL6ST, IL7R, INHBA, INPP4B, IRF2, IRF4, IRS2, ITGAV, ITGB1, ITK, ITPKB,JAK1, JAK2, JAK3, JAZF1, JUN, KAT6A, KAT6B, KCNJ5, KDM1A, KDM5A, KDM5C,KDM6A, KDR, KDSR, KEAP1, KEL, KIAA1549, KIF5B, KIR3DL1, KIT, KLF4,KLHL6, KLK2, KMT2A, KMT2C, KMT2D, KRAS, KTN1, LASP1, LCK, LCP1, LGALS3,LGR5, LHFP, LIFR, LMO1, LMO2, LOXL2, LPP, LRIG3, LRP1B, LUC7L2, LYL1,LYN, LZTR1, MAF, MAFB, MAGED1, MAGI2, MALT1, MAML2, MAP2K1, MAP2K2,MAP2K4, MAP3K1, MAPK1, MAPK11, MAX, MCL1, MDM2, MDM4, MDS2, MECOM,MED12, MEF2B, MEN1, MET, MITF, MKI67, MKL1, MLF1, MLH1, MLLT1, MLLT10,MLLT11, MLLT3, MLLT4, MLLT6, MMP9, MN1, MNX1, MPL, MRE11A, MS4A1, MSH2,MSH6, MSI2, MSN, MST1R, MTCP1, MTF2, MTOR, MUC1, MUC16, MUTYH, MYB, MYC,MYCL, MYCN, MYD88, MYH11, MYH9, NACA, NAE1, NBN, NCKIPSD, NCOA1, NCOA2,NCOA4, NDRG1, NF1, NF2, NFE2L2, NFIB, NFKB2, NFKBIA, NIN, NKX2-1, NONO,NOTCH1, NOTCH2, NOTCH3, NPM1, NR4A3, NRAS, NSD1, NT5C2, NTRK1, NTRK2,NTRK3, NUMA1, NUP214, NUP93, NUP98, NUTM1, NUTM2B, OLIG2, OMD, P2RY8,PAFAHIB2, PAK3, PALB2, PARK2, PARP1, PATZ1, PAX3, PAX5, PAX7, PAX8,PBRM1, PBX1, PCM1, PCSK7, PDCD1, PDCD1LG2, PDE4DIP, PDGFB, PDGFRA,PDGFRB, PDK1, PECAM1, PER1, PHF6, PHOX2B, PICALM, PIK3C2B, PIK3CA,PIK3CB, PIK3CD, PIK3CG, PIK3R1, PIK3R2, PIM1, PLAG1, PLCG2, PML, PMS1,PMS2, POLD1, POLE, POT1, POU2AF1, POU5F1, PPARG, PPP2R1A, PRCC, PRDM1,PRDM16, PREX2, PRF1, PRKAR1A, PRKCI, PRKDC, PRLR, PRPF40B, PRRT2, PRRX1,PRSS8, PSIP1, PSMD4, PTBP1, PTCH1, PTEN, PTK2, PTPN11, PTPRC, PTPRD,QKI, RABEP1, RAC1, RAD21, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAF1,RALGDS, RANBP17, RANBP2, RAP1GDS1, RARA, RB1, RBM10, RBM15, RCOR1,RECQL4, REL, RELN, RET, RHOA, RHOH, RICTOR, RIPK1, RMI2, RNF213, RNF43,ROS1, RPL10, RPL22, RPL5, RPN1, RPS6KB1, RPTOR, RUNX1, RUNX1T1, S1PR2,SAMHD1, SBDS, SDC4, SDHA, SDHAF2, SDHB, SDHC, SDHD, SEPT5, SEPT6, SEPT9,SET, SETBP1, SETD2, SF1, SF3A1, SF3B1, SF3B2, SFPQ, SGK1, SH2B3, SH3GL1,SLAMF7, SLC34A2, SLC45A3, SLIT2, SMAD2, SMAD3, SMAD4, SMARCA4, SMARCB1,SMARCE1, SMC1A, SMC3, SMO, SNCAIP, SNX29, SOCS1, SOX10, SOX11, SOX2,SOX9, SPECC1, SPEN, SPOP, SPTA1, SRC, SRGAP3, SRSF2, SRSF3, SS18,SS18L1, SSX1, STAG2, STAT3, STAT4, STAT5B, STEAP1, STIL, STK11, SUFU,SUZ12, SYK, TAF1, TAF15, TAL1, TAL2, TBL1XR1, TBX3, TCEA1, TCF12, TCF3,TCF7L2, TCL1A, TEK, TERC, TERT, TET1, TET2, TFE3, TFEB, TFG, TFPT, TFRC,TGFB1, TGFBR2, THRAP3, TIMP1, TJP1, TLX1, TLX3, TM7SF2, TMPRSS2,TNFAIP3, TNFRSF14, TNFRSF17, TNFRSF18, TNFRSF9, TNFSF11, TOP1, TOP2A,TP53, TP63, TPBG, TPM3, TPM4, TPR, TRAF2, TRAF3, TRAF3IP3, TRAF7,TRIM26, TRIM27, TRIM33, TRIP11, TRRAP, TSC1, TSC2, TSHR, TTK, TTL, TYMS,U2AF1, U2AF2, UBA1, UBR5, USP6, VEGFA, VEGFB, VHL, VPS51, VTI1A, WAS,WEE1, WHSC1, WHSC1L1, WIF1, WISP3, WNT11, WNT2B, WNT3, WNT3A, WNT4,WNTSA, WNT6, WNT7B, WRN, WT1, WWTR1, XBP1, XPA, XPC, XPO1, YWHAE, YWHAZ,ZAK, ZBTB16, ZBTB2, ZMYM2, ZMYM3, ZNF217, ZNF331, ZNF384, ZNF521, ZNF703and ZRSR2. As noted, a selection of genes can be assessed for copynumber variation. For example, the genes assessed by mutational analysisfor copy number variants may comprise at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or all of ABL1, AKT1, AKT2, ALK,ANG1/ANGPT1/TM7SF2, ANG2/ANGPT2NPS51, APC, ARAF, ARID1A, ATM, AURKA,AURKB, BBC3, BCL2, BIRC3, BRAF, BRCA1, BRCA2, CCND1, CCND3, CCNE1, CDK4,CDK6, CDK8, CDKN2A, CHEK1, CHEK2, CREBBP, CRKL, CSF1R, CTLA4, CTNNB1,DDR2, EGFR, EP300, ERBB3, ERBB4, EZH2, FBXW7, FGF10, FGF3, FGF4, FGFR1,FGFR2, FGFR3, FLT3, GATA3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2,JAK2, JAK3, KRAS, MCL1, MDM2, MLH1, MPL, MYC, NF1, NF2, NFKBIA, NOTCH1,NPM1, NRAS, NTRK1, PAX3, PAX5, PAX7, PAX8, PDGFRA, PDGFRB, PIK3CA,PTCH1, PTEN, PTPN11, RAF1, RB1, RET, RICTOR, ROS1, SMAD4, SRC, TOP1,TOP2A, TP53, VHL, and WT1. As noted, a selection of genes can beassessed for gene fusions. For example, the genes assessed by mutationalanalysis for fusion may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28or 29 of ALK, AR, BCR, BRAF, ETV1, ETV4, ETV5, ETV6, EWSR1, FGFR1,FGFR2, FGFR3, FUS, MYB, NFIB, NR4A3, NTRK1, NTRK2, NTRK3, PDGFRA, RAF1,RARA, RET, ROS1, SSX1, SSX2, SSX4, TFE3, and TMPRSS2.

In still other embodiments, the molecular profile comprises mutationalanalysis of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 of ALK, BRAF, BRCA1, BRCA2, EGFR, ERRB2, GNA11, GNAQ, IDH1,IDH2, KIT, KRAS, MET, NRAS, PDGFRA, PIK3CA, PTEN, RET, SRC and TP53. Themolecular profile may comprise mutational analysis of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27 or 28 of AKT1, HRAS, GNAS, MEK1, MEK2, ERK1, ERK2, ERBB3, CDKN2A,PDGFRB, IFG1R, FGFR1, FGFR2, FGFR3, ERBB4, SMO, DDR2, GRB1, PTCH, SHH,PD1, UGT1A1, BIM, ESR1, MLL, AR, CDK4 and SMAD4. The molecular profilemay also comprise mutational analysis of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 of ABL, APC, ATM, CDH1,CSFR1, CTNNB1, FBXW7, FLT3, HNF1A, JAK2, JAK3, KDR, MLH1, MPL, NOTCH1,NPM1, PTPN11, RB1, SMARCB1, STK11 and VHL. The genes assessed bymutational analysis may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 250, 300, or all genes, selected from the groupconsisting of ABL1, ABL2, ACVR1B, AKT1, AKT2, AKT3, ALK, AMER1(FAM123B), APC, AR, ARAF, ARFRP1, ARID1A, ARID1B, ARID2, ASXL1, ATM,ATR, ATRX, AURKA, AURKB, AXIN1, AXL, BAP1, BARD1, BCL2, BCL2L1, BCL2L2,BCL6, BCOR, BCORL1, BCR, BLM, BRAF, BRCA1, BRCA2, BRD4, BRIP1, BTG1,BTK, C11orf30 (EMSY), CARD11, CBFB, CBL, CCND1, CCND2, CCND3, CCNE1,CD274, CD79A, CD79B, CDC73, CDH1, CDK12, CDK4, CDK6, CDK8, CDKN1A,CDKN1B, CDKN2A, CDKN2B, CDKN2C, CEBPA, CHD2, CHD4, CHEK1, CHEK2, CIC,CREBBP, CRKL, CRLF2, CSF1R, CTCF, CTNNA1, CTNNB1, CUL3, CYLD, DAXX,DDR2, DICER1, DNMT3A, DOT1L, EGFR, EP300, EPHA3, EPHA5, EPHA7, EPHB1,ERBB2, ERBB3, ERBB4, ERG, ERRFI1, ESR1, ETV1, ETV4, ETV5, ETV6, EZH2,FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, FAS, FAT1,FBXW7, FGF10, FGF14, FGF19, FGF23, FGF3, FGF4, FGF6, FGFR1, FGFR2,FGFR3, FGFR4, FH, FLCN, FLT1, FLT3, FLT4, FOXL2, FOXP1, FRS2, FUBP1,GABRA6, GATA1, GATA2, GATA3, GATA4, GATA6, GID4 (C17orf39), GLI1, GNA11,GNA13, GNAQ, GNAS, GPR124, GRIN2A, GRM3, GSK3B, H3F3A, HGF, HNF1A, HRAS,HSD3B1, HSP90AA1, IDH1, IDH2, IGF1R, IGF2, IKBKE, IKZF1, IL7R, INHBA,INPP4B, IRF2, IRF4, IRS2, JAK1, JAK2, JAK3, JUN, KAT6A (MYST3), KDM5A,KDM5C, KDM6A, KDR, KEAP1, KEL, KIT, KLHL6, KMT2A (MLL), KMT2C (MLL3),KMT2D (MLL2), KRAS, LMO1, LRP1B, LYN, LZTR1, MAGI2, MAP2K1, MAP2K2,MAP2K4, MAP3K1, MCL1, MDM2, MDM4, MED12, MEF2B, MEN1, MET, MITF, MLH1,MPL, MRE11A, MSH2, MSH6, MTOR, MUTYH, MYB, MYC, MYCL (MYCL1), MYCN,MYD88, NF1, NF2, NFE2L2, NFKBIA, NKX2-1, NOTCH1, NOTCH2, NOTCH3, NPM1,NRAS, NSD1, NTRK1, NTRK2, NTRK3, NUP93, PAK3, PALB2, PARK2, PAX5, PBRM1,PDCD1LG2, PDGFRA, PDGFRB, PDK1, PIK3C2B, PIK3CA, PIK3CB, PIK3CG, PIK3R1,PIK3R2, PLCG2, PMS2, POLD1, POLE, PPP2R1A, PRDM1, PREX2, PRKAR1A, PRKCI,PRKDC, PRSS8, PTCH1, PTEN, PTPN11, QKI, RAC1, RAD50, RAD51, RAF1,RANBP2, RARA, RB1, RBM10, RET, RICTOR, RNF43, ROS1, RPTOR, RUNX1,RUNX1T1, SDHA, SDHB, SDHC, SDHD, SETD2, SF3B1, SLIT2, SMAD2, SMAD3,SMAD4, SMARCA4, SMARCB1, SMO, SNCAIP, SOCS1, SOX10, SOX2, SOX9, SPEN,SPOP, SPTA1, SRC, STAG2, STAT3, STAT4, STK11, SUFU, SYK, TAF1, TBX3,TERC, TERT, TET2, TGFBR2, TMPRSS2, TNFAIP3, TNFRSF14, TOP1, TOP2A, TP53,TSC1, TSC2, TSHR, U2AF1, VEGFA, VHL, WISP3, WT1, XPO1, ZBTB2, ZNF217,ZNF703. The mutational analysis may be performed to detect a generearrangement, e.g., a rearrangement in at least 1, e.g., at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28 or 29, of ALK, BCR, BCL2, BRAF, BRCA1, BRCA2,BRD4, EGFR, ETV1, ETV4, ETV5, ETV6, EWSR1, FGFR1, FGFR2, FGFR3, KIT,MSH2, MLL, MYB, MYC, NTRK1, NTRK2, PDGFRA, RAF1, RARA, RET, ROS1,TMPRSS2.

As noted, various cancers are characterized by chromosomaltranslocations and gene fusions. For example, acute lymphoblasticleukemia has been characterized by a number of kinase fusions. See, e.g,Table 16; G. Roberts et al., Targetable kinase-activating lesions inPh-like acute lymphoblastic leukemia. N. Engl. J. Med. 371, 1005-1015(2014), which reference is incorporated herein in its entirety.Crizotinib and imatinib target specific tyrosine kinases that formchimeric fusions. Crizotinib is FDA approved for ALK positive fusions inNSCLC and imatinib induces remission in leukemia patients that arepositive for BCR-ABL fusions. In an embodiment, the molecular profile ofthe invention comprises mutational analysis to assess a gene fusion inat least one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, ofABL1, ABL2, CSF1R, PDGFRB, CRLF2, JAK2, EPOR, IL2RB, NTRK3, PTK2B, TSLPand TYK2. Kinase fusions and other gene fusions have been observed in anumber of carcinomas. See, e.g., N. Stransky, E. Cerami, S. Schalm, J.L. Kim, C. Lengauer, The landscape of kinase fusions in cancer. NatCommun 5, 4846 (2014), which reference is incorporated herein in itsentirety. In another embodiment, mutational analysis is used to assess agene fusion in at least one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52 or 53, of AKT3, ALK, ARHGAP26, AXL, BRAF,BRD3, BRD4, EGFR, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EWSR1, FGFR1,FGFR2, FGFR3, FGR, INSR, MAML2, MAST1, MAST2, MET, MSMB, MUSK, MYB,NOTCH1, NOTCH2, NRG1, NTRK1, NTRK2, NTRK3, NUMBL, NUTM1, PDGFRA, PDGFRB,PIK3CA, PKN1, PPARG, PRKCA, PRKCB, RAF1, RELA, RET, ROS1, RSPO2, RSPO3,TERT, TFE3, TFEB, THADA and TMPRSS2. Fusions with any desired number ofthese genes can be detected in carcinomas of various lineages.Similarly, a number of gene fusions have been detected in a variety ofsarcomas. In an embodiment, mutational analysis is used to assess a genefusion in at least one, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26, ofALK, CAMTA1, CCNB3, CIC, EPC, EWSR1, FKHR, FUS, GLI1, HMGA2, JAZF1,MEAF6, MKL2, NCOA2, NTRK3, PDGFB, PLAG1, ROS1, SS18, STATE, TAF15,TCF12, TFE3, TFG, USP6 and YWHAE. Any desired number of fusions in thesegenes can be detected in various sarcomas. Additional gene fusions thatcan be detected as part of the molecular profiling of the invention aredescribed in M. J. Annala, B. C. Parker, W. Zhang, M. Nykter, Fusiongenes and their discovery using high throughput sequencing. Cancer Lett.340, 192-200 (2013), which reference is incorporated herein in itsentirety. Gene fusions can be detected by various technologies,including without limitation IHC (e.g., to detect mutant proteinsproduced by gene fusions), ISH, PCR (e.g., RT-PCR), microarrays andsequencing analysis. In an embodiment, the fusions are detected usingNext Generation Sequencing technology.

TABLE 16 Kinase gene fusions Kinase Gene 5′ Genes ABL1 ETV6, NUP214,RCSD1, RANBP2, SNX2, ZMIZ1 ABL2 PAG1, RCSD1 CSF1R SSBP2 PDGFRB EBF1,SSBP2, TNIP1, ZEB2 CRLF2 P2RY8 JAK2 ATF7IP, BCR, ETV6, PAX5, PPFIBP1,SSBP2, STRN3, TERF2, TPR EPOR IGH, IGK IL2RB MYH9 NTRK3 ETV6 PTK2BKDM6A, STAG2 TSLP IQGAP2 TYK2 MYB

Various cancer genes disclosed in the COSMIC (Catalogue Of SomaticMutations In Cancer) database (available atcancer.sanger.ac.uk/cancergenome/projects/cosmic/) can be assessed aswell.

Lab Technique Substitution

One of skill will appreciate that the laboratory techniques of themolecular profiles herein can be substituted by alternative techniquesif appropriate, including alternative techniques as disclosed herein orknown in the art. For example, FISH and CISH are generallyinterchangeable methods so that one can often be used in place of theother. Similarly, Dual ISH methods such as described herein can besubstituted for conventional ISH methods. In an embodiment, the FDAapproved INFORM HER2 Dual ISH DNA Probe Cocktail kit from VentanaMedical Systems, Inc. (Tucson, Ariz.) is used for FISH/CISH analysis ofHER2. This kit allows the determination of the HER2 gene status byenumeration of the ratio of the HER2 gene to Chromosome 17. The HER2 andChromosome 17 probes are detected using two color chromogenic in situhybridization (CISH) reactions. A number of methods can be used toassess nucleic acid sequences, and any alterations thereof, includingwithout limitation point mutations, insertions, deletions,translocations, rearrangements. Nucleic acid analysis methods includeSanger sequencing, next generation sequencing, polymerase chain reaction(PCR), real-time PCR (qPCR; RT-PCR), a low density microarray, a DNAmicroarray, a comparative genomic hybridization (CGH) microarray, asingle nucleotide polymorphism (SNP) microarray, fragment analysis,RFLP, pyrosequencing, methylation specific PCR, mass spec, Southernblotting, hybridization, and related methods such as described herein.Similarly, a number of methods can be used to assess gene expression,including without limitation next generation sequencing, polymerasechain reaction (PCR), real-time PCR (qPCR; RT-PCR), a low densitymicroarray, a DNA microarray, a comparative genomic hybridization (CGH)microarray, a single nucleotide polymorphism (SNP) microarray, proteomicarrays, antibody arrays or mass spec. The presence or level of a proteincan also be assessed using multiple methods as appropriate, includingwithout limitation IHC, immunocapture, immunoblotting, Western analysis,ELISA, immunoprecipitation, flow cytometry, and the like. The desiredlaboratory technique can be chosen based of multiple criteria, includingwithout limitation accuracy, precision, reproduceability, cost, amountof sample available, type of sample available, time to perform thetechnique, regulatory approval status of the technique platform,regulatory approval status of the particular test, and the like.

In some embodiments, more than one technique is used to assess a samebiomarker. For example, results of profiling both gene expression andprotein expression can provide confirmatory results. In other cases, acertain method may provide optimal results depending on the availablesample. In some embodiments, sequencing is used to assess EGFR if thesample is more than 50% tumor. Fragment analysis (FA) can also be usedto assess EGFR. In some embodiments, FA, e.g., RFLP, is used to assessEGFR if the sample is less than 50% tumor. In still other cases, onetechnique may indicate a desire to perform another technique, e.g., aless expensive technique or one that requires lesser sample quantity mayindicate a desire to perform a more expensive technique or one thatconsumes more sample. In an embodiment, FA of ALK is performed first,and then FISH or PCR is performed if the FA indicates the presence of aparticular ALK alteration such as an ALK fusion. The FISH and/or PCRassay can be designed such that only certain fusion products aredetected, e.g., EML4-ALK. The alternate methods may also providedifferent information about the biomarker. For example, sequenceanalysis may reveal the presence of a mutant protein, whereas IHC of theprotein may reveal its level and/or cellular location. As anotherexample, gene copy number or gene expression at the RNA level may beelevated, but the presence of interfering RNAs may still downregulateprotein expression. As still another example, a biomarker can beassessed using a same technique but with different reagents that provideactionable results. As an example, SPARC can be assessed by IHC usingeither a polyclonal or a monoclonal antibody. This context is identifiedherein, e.g., as SPARCp, SPARC poly, or variants thereof for SPARCdetected using a polyclonal antibody), and as SPARCm, SPARC mono, orvariants thereof, for SPARC detected using a monoclonal antibody). SPARC(m/p) and similar derivations can be used to refer to IHC performedusing both polyclonal and monoclonal antibodies.

One of skill will appreciate that molecular profiles of the inventioncan be updated as new evidence becomes available. For example, newevidence may appear in the literature describing an association betweena treatment and potential benefit for cancer or a certain lineage ofcancer. This information can be incorporated into an appropriatemolecular profile. As another example, new evidence may be presented fora biomarker that is already assessed according to the invention.Consider the BRAF V600E mutation that is currently FDA approved fordirected treatment with vemurafenib for melanoma. If the treatment isdetermined to be effective in another setting, e.g., for another lineageof cancer, BRAF V600E can be added to an appropriate molecular profilefor that setting.

Clinical Trial Connector

Thousands of clinical trials for therapies are underway in the UnitedStates, with several hundred of these tied to biomarker status. In anembodiment, the molecular intelligence molecular profiles of theinvention include molecular profiling of markers that are associatedwith ongoing clinical trials. Thus, the molecular profile can be linkedto clinical trials of therapies that are correlated to a subject'sbiomarker profile. The method can further comprise identifying triallocation(s) to facilitate patient enrollment. The database of ongoingclinical trials can be obtained from www.clinicaltrials.gov in theUnited States, or similar source in other locations. The molecularprofiles generated by the methods of the invention can be linked toongoing clinical trials and updated on a regular basis, e.g., daily,bi-weekly, weekly, monthly, or other appropriate time period.

Although significant advances in cancer treatment have been made inrecent years, not all patients can be effectively treated within thestandard of care paradigm. Many patients are eligible for clinicaltrials participation, yet less than 3 percent are actually enrolled in atrial, according to recent National Cancer Institute (NCI) statistics.The Clinical Trials Connector allows caregivers such as physicians toquickly identify and review global clinical trial opportunities inreal-time that are molecularly targeted to each patient. In embodiments,the Clinical Trials Connector has one or more of the following features:Examines thousands of open and enrolling clinical trials; Individualizesclinical trials based on molecular profiling as described herein;Includes interactive and customizable trial search filters by:Biomarker, Mechanism of action, Therapy, Phase of study, and otherclinical factors (age, sex, etc.). The Clinical Trials Connector can bea computer database that is accessed once molecular profiling resultsare available. In some embodiments, the database comprises theEmergingMed database (EmergingMed, New York, N.Y.).

Tables 6 and Tables 9-10 herein indicates an association of certainbiomarkers in the molecular profiles of the invention with ongoingclinical trials. Profiling of the specified markers can provide anindication that a subject is a candidate for a clinical trial, e.g., bysuggesting that an agent in a clinical trial may benefit the subject.For example, Table 9 indicates that molecular profiling of the followingbiomarkers may provide an indication that an individual meets inclusioncriteria for an ongoing clinical trial: EGFR or PTEN by IHC; detectionof EGFR vIII (e.g., by fragment analysis); MET by ISH or sequenceanalysis (e.g., NGS), MLH1, MSH2, MSH6, PMS2 by IHC or dection of MSI(e.g., by fragment analysis); and/or mutational analysis of at least oneof ABL1, AKT1, ALK, APC, ATM, CSF1R, CTNNB1, EGFR, ERBB2 (Her2), FGFR1,FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR (VEGFR2), KRAS,MPL, NOTCH1, NRAS, PTEN, SMO, TP53, VHL, and any combination thereof(e.g., by NGS). One of skill can identify appropriate clinical trials,e.g., by searching www.clinicaltrials.gov by the various biomarkers ofinterest and determining whether the molecular profiling resultsindicated the patient meets eligibility criteria for the identifiedtrials.

In an aspect, the invention provides a set of rules for matching ofclinical trials to biomarker status as determined by the molecularprofiling described herein. In some embodiments, the matching ofclinical trials to biomarker status is performed using one or morepre-specified criteria: 1) Trials are matched based on the OFF NCCNCompendia drug/drug class associated with potential benefit by themolecular profiling rules; 2) Trials are matched based on biomarkerdriven eligibility requirement of the trial; and 3) Trials are matchedbased on the molecular profile of the patient, the biology of thedisease and the associated signaling pathways. In the latter case, i.e.item 3, clinical trial matching may comprise further criteria asfollows. First, for directly targetable markers, match trials withagents directly targeting the gene (e.g., FGFR results map to anti-FGFRtherapy trials; ERBB2 results map to anti-HER2 agents, etc). Inaddition, for directly targetable markers, trial matching considersdownstream markers under the following scenarios: a) a known resistancemechanism is available (e.g., cMET inhibitors for EGFR gene); b)clinical evidence associates the (mutated) biomarker with drugstargeting downstream pathways (e.g., mTOR inhibitors when PIK3CA ismutated); and c) active clinical trials are enrolling patients (with thebiomarker aberration in the inclusion criteria) with drugs targeting thedownstream pathways (e.g., SMO inhibitors for BCR-ABL mutation T315I).In the case of markers that are not directly targetable by a knowntherapeutic agent, trial matching may consider alternative, downstreammarkers (e.g., platinum agents for ATM gene; MEK inhibitors forGNAS/GNAQ/GNA11 mutation). The clinical trials that are matched may beidentified based on results of “pathogenic,” “presumed pathogenic,” orvariant of uncertain (or unknown) significance (“VUS”). In someembodiments, the decision to incorporate/associate a drug class with abiomarker mutation can further depend on one or more of thefollowing: 1) Clinical evidence; 2) Preclinical evidence; 3)Understanding of the biological pathway affected by the biomarker; and4) expert analysis. In some embodiments, the mutation of biomarkers inthe above section “Mutational Analysis” is linked to clinical trialsusing one or more of these criteria.

The guiding principle above can be used to identify classes of drugsthat are linked to certain biomarkers. The biomarkers can be linked tovarious clinical trials that are studying these biomarkers, includingwithout limitation requiring a certain biomarker status for clinicaltrial inclusion. Clinical trials studying the drug classes and/orspecific agents listed can be matched to the biomarker. In an aspect,the invention provides a method of selecting a clinical trial forenrollment of a patient, comprising performing molecular profiling ofone or more biomarker on a sample from the patient using the methodsdescribed herein. For example, the profiling can be performed for one onmore biomarker in any of Tables 6-10 or 12-15 using the techniqueindicated in the table. The results of the profiling are matched toclasses of drugs using the above criteria. Clinical trials studyingmembers of the classes of drugs are identified. The patient is apotential candidate for the so-identified clinical trials.

Report

In an embodiment, the methods of the invention comprise generating amolecular profile report. The report can be delivered to the treatingphysician or other caregiver of the subject whose cancer has beenprofiled. The report can comprise multiple sections of relevantinformation, including without limitation: 1) a list of the genes and/orgene products in the molecular profile; 2) a description of themolecular profile of the genes and/or gene products as determined forthe subject; 3) a treatment associated with one or more of the genesand/or gene products in the molecular profile; and 4) and an indicationwhether each treatment is likely to benefit the patient, not benefit thepatient, or has indeterminate benefit. The list of the genes and/or geneproducts in the molecular profile can be those presented herein for themolecular intelligence profiles of the invention. The description of themolecular profile of the genes and/or gene products as determined forthe subject may include such information as the laboratory techniqueused to assess each biomarker (e.g., RT-PCR, FISH/CISH, IHC, PCR,FA/RFLP, sequencing, etc) as well as the result and criteria used toscore each technique. By way of example, the criteria for scoring aprotein as positive or negative for IHC may comprise the amount ofstaining and/or percentage of positive cells, the criteria for scoring anucleic acid RT-PCR may be a cycle number indicating whether the levelof the appropriate nucleic acid is differentially regulated as comparedto a control sample, or criteria for scoring a mutation may be apresence or absence. The treatment associated with one or more of thegenes and/or gene products in the molecular profile can be determinedusing a biomarker-drug association rule set as described herein, e.g.,in any one of Tables 3-6, Tables 9-10, Table 17, and Tables 22-24. Theindication whether each treatment is likely to benefit the patient, notbenefit the patient, or has indeterminate benefit may be weighted. Forexample, a potential benefit may be a strong potential benefit or alesser potential benefit. Such weighting can be based on any appropriatecriteria, e.g., the strength of the evidence of the biomarker-treatmentassociation, or the results of the profiling, e.g., a degree of over- orunderexpression.

Various additional components can be added to the report as desired. Inan embodiment, the report comprises a list having an indication ofwhether one or more of the genes and/or gene products in the molecularprofile are associated with an ongoing clinical trial. The report mayinclude identifiers for any such trials, e.g., to facilitate thetreating physician's investigation of potential enrollment of thesubject in the trial. In some embodiments, the report provides a list ofevidence supporting the association of the genes and/or gene products inthe molecular profile with the reported treatment. The list can containcitations to the evidentiary literature and/or an indication of thestrength of the evidence for the particular biomarker-treatmentassociation. In still another embodiment, the report comprises adescription of the genes and/or gene products in the molecular profile.The description of the genes and/or gene products in the molecularprofile may comprise without limitation the biological function and/orvarious treatment associations.

FIGS. 27A-X herein presents an illustrative patient report according tothe invention. The illustrative report was derived from molecularprofiling of a triple negative breast cancer with mutational analysisusing an expanded Next Generation sequencing panel as described herein(see, e.g., Tables 8 and 12-15).

As noted herein, the same biomarker may be assessed by one or moretechnique. In such cases, the results of the different analysis may beprioritized in case of inconsistent results. For example, the differentmethods may detect different aspects of a single biomarker (e.g.,expression level versus mutation), or one method may be more sensitivethan another. In one example, consider that molecular profiling resultsobtained using the FDA approved cobas PCR (Roche Diagnostics) can beprioritized over Next Generation sequencing results. However, if thesequencing detects a mutation, e.g., V600E, V600E2 or V600K, when PCReither detects wild type or is not determinable, the report may containa note describing both sets of results including any therapy that may beimplicated. In the case of melanoma, when the result of BRAF cobas PCRis “Wild type” or “no data” whereas BRAF sequencing is “V600E” or“V600E2”, the report may comprise a note that BRAF mutation was notdetected by the FDA-approved Cobas PCR test, however, a V600E/E2mutation was detected by alternative methods (next generation/Sangersequencing) and that evidence suggests that the presence of a V600Emutation associates with potential clinical benefit from vemurafenib,dabrafenib or trametinib therapy. Similarly, when the result of BRAFcobas PCR is “Wild type” or “no data” and BRAF sequencing is “V600K”,the report may comprise a note that BRAF mutation was not detected bythe FDA-approved Cobas PCR test, however, a V600K mutation was detectedby alternative methods (next generation/Sanger sequencing) and thatevidence suggests that the presence of a V600K mutation associates withpotential clinical benefit from trametinib therapy.

The molecular profiling report can be delivered to the caregiver for thesubject, e.g., the oncologist or other treating physician. The caregivercan use the results of the report to guide a treatment regimen for thesubject. For example, the caregiver may use one or more treatmentsindicated as likely benefit in the report to treat the patient.Similarly, the caregiver may avoid treating the patient with one or moretreatments indicated as likely lack of benefit in the report.

Immune Modulators

PD1 (programmed death-1, PD-1) is a transmembrane glycoprotein receptorthat is expressed on CD4-/CD8-thymocytes in transition to CD4+/CD8+stage and on mature T and B cells upon activation. It is also present onactivated myeloid lineage cells such as monocytes, dendritic cells andNK cells. In normal tissues, PD-1 signaling in T cells regulates immuneresponses to diminish damage, and counteracts the development ofautoimmunity by promoting tolerance to self-antigens. PD-L1 (programmedcell death 1 ligand 1, PDL1, cluster of differentiation 274, CD274, B7homolog 1, B7-H1, B7H1) and PD-L2 (programmed cell death 1 ligand 2,PDL2, B7-DC, B7DC, CD273, cluster of differentiation 273) are PD1ligands. PD-L1 is constitutively expressed in many human cancersincluding without limitation melanoma, ovarian cancer, lung cancer,clear cell renal cell carcinoma (CRCC), urothelial carcinoma, HNSCC, andesophageal cancer. Blockade of PD-1 which is expressed intumor-infiltrating T cells (TILs) has created an important rationale fordevelopment to monoclonal antibody therapy to target blockade ofPD1/PDL-1 pathway. Tumor cell expression of PD-L1 is used as a mechanismto evade recognition/destruction by the immune system as in normal cellsthe PD1/PDL1 interplay is an immune checkpoint. Monoclonal antibodiestargeting PD-1/PD-L1 that boost the immune system are being developedfor the treatment of cancer. See, e.g., Flies et al, Blockade of theB7-H1/PD-1 pathway for cancer immunotherapy. Yale J Biol Med. 2011December; 84(4):409-21; Sznol and Chen, Antagonist Antibodies to PD-1and B7-H1 (PD-L1) in the Treatment of Advanced Human Cancer, Clin CancerRes; 19(5) Mar. 1, 2013; Momtaz and Postow, Immunologic checkpoints incancer therapy: focus on the programmed death-1 (PD-1) receptor pathway.Pharmgenomics Pers Med. 2014 Nov. 15; 7:357-65; Shin and Ribas, Theevolution of checkpoint blockade as a cancer therapy: what's here,what's next?, Curr Opin Immunol. 2015 Jan. 23; 33C:23-35; whichreferences are incorporated by reference herein in their entirety.Several drugs are in clinical development that affect the PDL1/PD1pathway include: 1) Nivolumab (BMS936558/MDX-1106), an anti-PD1 drugfrom Bristol Myers Squib drug which was approved by the U.S. FDA in late2014 under the brand name OPDIVO for the treatment of patients withunresectable or metastatic melanoma and disease progression followingipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor; 2)Pembrolizumab (formerly lambrolizumab, MK-3475, trade name Keytruda), ananti-PD1 drug from Merck approved in late 2014 for use followingtreatment with ipilimumab, or after treatment with ipilimumab and a BRAFinhibitor in patients who carry a BRAF mutation; 3) BMS-936559/MDX-1105,an anti-PDL1 drug from Bristol Myers Squib with initial evidence inadvanced solid tumors; and 4) MPDL3280A, an anti-PDL1 drug from Rochewith initial evidence in NSCLC.

Expression of PD1, PD-L1 and/or PD-L2 expression can be assessed at theprotein and/or mRNA level according to the methods of the invention. Forexample, IHC can be used to assess their protein expression. Expressionmay indicate likely benefit of inhibitors of the B7-H1/PD-1 pathway,whereas lack of expression may indicate lack of benefit thereof. In someembodiments, expression of both PD-1 and PD-L1 is assessed and likelybenefit of inhibitors of the B7-H1/PD-1 pathway is determined only uponco-expression of both of these immunosuppressive components. Certaincells express PD-L1 mRNA, but not the protein, due to translationalsuppression by microRNA miR-513. Therefore, analysis of PD-L1 proteinmay be desirable for molecular profiling. Molecular profiling may alsoinclude that of miR-513. Expression of miR-513 above a certain thresholdmay indicate lack of benefit of immune modulation therapy.

In an aspect, the invention provides a method of identifying at leastone treatment associated with a cancer in a subject, comprising: a)determining a molecular profile for at least one sample from the subjectby assessing a plurality of gene or gene products, wherein the pluralityof genes and/or gene products comprises at least one of PD-1 and PD-L1;and b) identifying, based on the molecular profile, at least one of: i)at least one treatment that is associated with benefit for treatment ofthe cancer; ii) at least one treatment that is associated with lack ofbenefit for treatment of the cancer; and iii) at least one treatmentassociated with a clinical trial. Expression of PD-1 and/or PD-L1 may beperformed along with that of additional biomarkers that guide treatmentselection according to the invention. Such additional biomarkers can beadditional immune modulators including without limitation CTL4A, IDO1,COX2, CD80, CD86, CD8A, Granzyme A, Granzyme B, CD19, CCR7, CD276,LAG-3, TIM-3, and a combination thereof. The additional biomarkers couldalso comprise other useful biomarkers disclosed herein, such any ofTables 2, 6, or 12-15. For example, the additional biomarkers maycomprise at least one of 1p19q, ABL1, AKT1, ALK, APC, AR, ATM, BRAF,BRCA1, BRCA2, cKIT, cMET, CSF1R, CTNNB1, EGFR, EGFRvIII, ER, ERBB2(HER2), FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HRAS, IDH1, IDH2,JAK2, KDR (VEGFR2), KRAS, MGMT, MGMT-Me, MLH1, MPL, NOTCH1, NRAS,PDGFRA, Pgp, PIK3CA, PR, PTEN, RET, RRM1, SMO, SPARC, TLE3, TOP2A,TOPO1, TP53, TS, TUBB3, VHL, CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1,PTPN11, RB1, SMAD4, SMARCB1, STK1, MLH1, MSH2, MSH6, PMS2,microsatellite instability (MSI), ROS1 and ERCC1. These additionalanalyses may suggest combinations of therapies likely to benefit thepatient, such as a PD-1/PD-L1 pathway inhibitor and another therapysuggested by the molecular profiling. See, e.g., additionalbiomarker-drug associations in any of Tables 3-6, Tables 9-10, Table 17,and Tables 22-24. In some embodiments, anti-CTLA-4 therapy, includingwithout limitation ipilimumab, is administered with PD-1/PD-L1 pathwaytherapy.

The invention further provides association of immune modulation therapy,including without limitation PD-1/PD-L1 pathway inhibitor treatments,with molecular profiling of biomarkers in addition to PD-1/PD-L1themselves. In an embodiment of the invention, beneficial treatment ofthe cancer with immunotherapy targeting at least one of PD-1, PD-L1,CTLA-4, IDO-1, and CD276, is associated with a molecular profileindicating that the cancer is AR−/HER2−/ER−/PR− (quadruple negative)and/or carries a mutation in BRCA1. In some embodiments, the inventionprovides associating beneficial treatment of the cancer withimmunotherapy targeting immune modulating therapy wherein the molecularprofile indicates that the cancer carries a mutation in at least onecancer-related gene. The cancer-related gene can include at least one,e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47, of ABL1, AKT1,ALK, APC, ATM, BRAF, BRCA1, BRCA2, cKIT, cMET, CSF1R, CTNNB1, EGFR,ERBB2, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, JAK2, KDR(VEGFR2), KRAS, MLH1, MPL, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, SMO,TP53, VHL, CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4,SMARCB1 and STK1. Other cancer related genes, such as those disclosedherein or in the COSMIC (Catalogue Of Somatic Mutations In Cancer)database (available atcancer.sanger.ac.uk/cancergenome/projects/cosmic/), can be assessed aswell. See Example 10 herein. It will be apparent to one of skill thatsuch profiling may be performed independently of direct assessment ofimmune modulators themselves. As an illustrative example, a tumordetermined to carry a mutation in BRCA1 may be a candidate for anti-PD-1and/or anti-PD-L1 therapy. Thus, in a related aspect, the inventionprovides a method of identifying at least one treatment associated witha cancer in a subject, comprising: a) determining a molecular profilefor at least one sample from the subject by assessing a plurality ofgenes and/or gene products other than PD-1 and/or PD-L1; and b)identifying, based on the molecular profile, that the cancer is likelyto benefit from anti-PD-1 or anti-PD-L1 therapy.

Expression of PD-1 is generally assessed in tumor infiltratinglymphocytes (TILs). PD-L1 may be expressed in various cells in the tumormicroenvironment. In addition to tumor cells, PD-L1 can be expressed byT cells, natural killer (NK) cells, macrophages, myeloid dendritic cells(DCs), B cells, epithelial cells, and vascular endothelial cells. Insome cases, the response to anti-PD-1/PD-L1 therapy may be dependent onwhich cells in the tumor microenvironment express PD-L1. Thus, in someembodiments of the invention, the tumor microenvionment is assessed todetermine the expression patterns of PD-L1 and the likely benefit orlack thereof is dependent on the cells determined to express PD-L1. SuchPD-L1 expression can be determined in various cells, including withoutlimitation one or more of T cells, natural killer (NK) cells,macrophages, myeloid dendritic cells (DCs), B cells, epithelial cells,and endothelial cells.

Certain tumor cells may also more susceptible to immune modulatingtherapy and thus more likely associated with likely treatment benefit.An “immune modulating therapy” can include antagonists such asantibodies to PD-1, PD-L1, PD-L2, CTL4A, IDOL COX2, CD80, CD86, CD8A,Granzyme A, Granzyme B, CD19, CCR7, CD276, LAG-3 or TIM-3. Theantagonist could also be a soluble ligand or small molecule inhibitor.As a non-limiting example, a soluble PD-L1 construct may bind PD-1 andthus block its immunosuppressive activity. In an embodiment, theinvention provides for determining the apoptotic or necrotic environmentof the tumor. Apoptotic or necrotic cells may be associated with likelytreatment benefit from immune modulating therapy. Thus, the inventionprovides a method of identifying at least one treatment associated witha cancer in a subject, comprising: a) determining a molecular profilefor at least one sample from the subject by assessing tumor necrosis orapoptosis; and b) associating the cancer with likely to benefit fromimmune modulating therapy, including without limitation anti-PD-1 oranti-PD-L1 therapy, if apoptotic or necrotic tumor cells are identified.

EXAMPLES Example 1: Molecular Profiling to Find Targets and SelectTreatments for Refractory Cancers

The primary objective was to compare progression free survival (PFS)using a treatment regimen selected by molecular profiling with the PFSfor the most recent regimen the patient progressed on (e.g. patients aretheir own control) (FIG. 28). The molecular profiling approach wasdeemed of clinical benefit for the individual patient who had a PFSratio (PFS on molecular profiling selected therapy/PFS on prior therapy)of ≥1.3.

The study was also performed to determine the frequency with whichmolecular profiling by IHC, FISH and microarray yielded a target againstwhich there is a commercially available therapeutic agent and todetermine response rate (RECIST) and percent of patients withoutprogression or death at 4 months.

The study was conducted in 9 centers throughout the United States. Anoverview of the method is depicted in FIG. 29. As can be seen in FIG.29, the patient was screened and consented for the study. Patienteligibility was verified by one of two physician monitors. The samephysicians confirmed whether the patients had progressed on their priortherapy and how long that PFS (TTP) was. A tumor biopsy was thenperformed, as discussed below. The tumor was assayed using IHC, FISH (onparaffin-embedded material) and microarray (on fresh frozen tissue)analyses.

The results of the IHC/FISH and microarray were given to two studyphysicians who in general used the following algorithm in suggestingtherapy to the physician caring for the patient: 1) IHC/FISH andmicroarray indicated same target was first priority; 2) IHC positiveresult alone next priority; and 3) microarray positive result alone thelast priority.

The patient's physician was informed of the suggested treatment and thepatient was treated with the suggested agent(s) (package insertrecommendations). The patient's disease status was assessed every 8weeks and adverse effects were assessed by the NCI CTCAE version 3.0.

To be eligible for the study, the patient was required to: 1) provideinformed consent and HIPAA authorization; 2) have any histologic type ofmetastatic cancer; 3) have progressed by RECIST criteria on at least 2prior regimens for advanced disease; 4) be able to undergo a biopsy orsurgical procedure to obtain tumor samples; 5) be ≥18 years, have a lifeexpectancy >3 months, and an Eastern Cooperative Oncology Group (ECOG)Performance Status or 0-1; 6) have measurable or evaluable disease; 7)be refractory to last line of therapy (documented disease progressionunder last treatment; received ≥6 weeks of last treatment; discontinuedlast treatment for progression); 8) have adequate organ and bone marrowfunction; 9) have adequate methods of birth control; and 10) if CNSmetastases then adequately controlled. The ECOG performance scale isdescribed in Oken, M. M., Creech, R. H., Tormey, D. C., Horton, J.,Davis, T. E., McFadden, E. T., Carbone, P. P.: Toxicity And ResponseCriteria Of The Eastern Cooperative Oncology Group. Am J Clin Oncol5:649-655, 1982, which is incorporated by reference in its entirety.Before molecular profiling was performed, the principal investigator atthe site caring for the patient must designate what they would treat thepatient with if no molecular profiling results were available.

Methods

All biopsies were performed at local investigators' sites. For needlebiopsies, 2-3 18 gauge needle core biopsies were performed. For DNAmicroarray (MA) analysis, tissue was immediately frozen and shipped ondry ice via FedEx to a central CLIA certified laboratory, Caris MPI inPhoenix, Ariz. For IHC, paraffin blocks were shipped on cold packs. IHCwas considered positive for target if 2+ in ≥30% of cells. The MA wasconsidered positive for a target if the difference in expression for agene between tumor and control organ tissue was at a significance levelof p≤0.001.

Ascertainment of the Time to Progression to Document theProgression-Free Survival Ratio

Time to progression under the last line of treatment was documented byimaging in 58 patients (88%). Among these 58 patients, documentation byimaging alone occurred in 49 patients (74%), and documentation byimaging with tumor markers occurred in nine patients (14%; ovariancancer, n 3; colorectal, n 1; pancreas, n 1; prostate, n 3; breast, n1). Patients with clinical proof of progression were accepted when theinvestigator reported the assessment of palpable and measurable lesions(i.e., inflammatory breast cancer, skin/subcutaneous nodules, or lymphnodes), which occurred in six patients (9%). One patient (2%) withprostate cancer was included with progression by tumor marker. In onepatient (2%) with breast cancer, the progression was documented byincrease of tumor marker and worsening of bone pain. The time toprogression achieved with a treatment based on molecular profiling wasdocumented by imaging in 44 patients (67%) and by clinical eventsdetected between two scheduled tumor assessments in 20 patients. Theseclinical events were reported as serious adverse events related todisease progression (e.g., death, bleeding, bowel obstruction,hospitalization), and the dates of reporting were censored asprogression of disease. The remaining two patients were censored at thedate of last follow-up.

IHC/FISH

For IHC studies, the formalin fixed, paraffin embedded tumor samples hadslices from these blocks submitted for IHC testing for the followingproteins: EGFR, SPARC, C-kit, ER, PR, Androgen receptor, PGP, RRM1,TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, Thymidylate synthase,Her2/neu and TOPO2A. IHCs for all proteins were not carried out on allpatients' tumors.

Formalin-fixed paraffin-embedded patient tissue blocks were sectioned (4μm thick) and mounted onto glass slides. After deparaffination andrehydration through a series of graded alcohols, pretreatment wasperformed as required to expose the targeted antigen.

Human epidermal growth factor receptor 2 (HER2) and epidermal growthfactor receptor (EGFR) were stained as specified by the vendor (DAKO,Denmark). All other antibodies were purchased from commercial sourcesand visualized with a DAB biotin-free polymer detection kit. Appropriatepositive control tissue was used for each antibody. Negative controlslides were stained by replacing the primary antibody with anappropriately matched isotype negative control reagent. All slides werecounterstained with hematoxylin as the final step and cover slipped.Tissue microarray sections were analyzed by FISH for EGFR and HER-2/neucopy number per the manufacturer's instructions. FISH for HER-2/neu (wasdone with the PathVysion HER2 DNA Probe Kit (Abbott Molecular, AbbottPark, Ill.). FISH for EGFR was done with the LSI EGFR/CEP 7 Probe(Abbott Molecular).

All slides were evaluated semi-quantitatively by a first pathologist,who confirmed the original diagnosis as well as read each of theimmunohistochemical stains using a light microscope. Some lineageimmunohistochemical stains were performed to confirm the originaldiagnosis, as necessary. Staining intensity and extent of staining weredetermined; both positive, tumor-specific staining of tumor cells andhighly positive (≥2+), pervasive (≥30%) tumor specific staining resultswere recorded. IHC was considered positive for target if staining was≥2+ in ≥30% of cells. Rather than look for a positive signal withoutqualification, this approach raises the stringency of the cut point suchthat it would be a significant or more demonstrative positive. A higherpositive is more likely to be associated with a therapy that wouldaffect the time to progression. The cut point used (i.e., staining was≥2+ in ≥30% of cells) is similar to some cut points used in breastcancer for HER2/neu. When IHC cut points were compared with evidencefrom the tissue of origin of the cancer, the cut points were equal to orhigher (more stringent) than the evidence cut points. A standard 10%quality control was performed by a second pathologist.

Microarray

Tumor samples obtained for microarray were snap frozen within 30 minutesof resection and transmitted to Caris-MPI on dry ice. The frozen tumorfragments were placed on a 0.5 mL aliquot of frozen 0.5M guanidineisothiocyanate solution in a glass tube, and simultaneously thawed andhomogenized with a Covaris S2 focused acoustic wave homogenizer(Covaris, Woburn, Mass.). A 0.5 mL aliquot of TriZol was added, mixedand the solution was heated to 65° C. for 5 minutes then cooled on iceand phase separated by the addition of chloroform followed bycentrifugation. An equal volume of 70% ethanol was added to the aqueousphase and the mixture was chromatographed on a Qiagen RNeasy column(Qiagen, Germantown, Md.). RNA was specifically bound and then eluted.The RNA was tested for integrity by assessing the ratio of 28S to 18Sribosomal RNA on an Agilent BioAnalyzer (Agilent, Santa Clara, Calif.).Two to five micrograms of tumor RNA and two to five micrograms of RNAfrom a sample of a normal tissue representative of the tumor's tissue oforigin were separately converted to cDNA and then labeled during T7polymerase amplification with contrasting fluor tagged (Cy3, Cy5)cytidine triphosphate. The labeled tumor and its tissue of originreference were hybridized to an Agilent H1Av2 60-mer olio array chipwith 17,085 unique probes.

The arrays contain probes for 50 genes for which there is a possibletherapeutic agent that would potentially interact with that gene (witheither high expression or low expression). Those 50 genes included: ADA,AR, ASNA, BCL2, BRCA2, CD33, CDW52, CES2, DNMT1, EGFR, ERBB2, ERCC3,ESR1, FOLR2, GART, GSTP1, HDAC1, HIF1A, HSPCA, IL2RA, KIT, MLH1, MS4A1,MASH2, NFKB2, NFKBIA, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN,PTGS2, RAF1, RARA, RXRB, SPARC, SSTR1, TK1, TNF, TOP1, TOP2A, TOP2B,TXNRD1, TYMS, VDR, VEGF, VHL, and ZAP70.

The chips were hybridized from 16 to 18 hours at 60° C. and then washedto remove non-stringently hybridized probe and scanned on an AgilentMicroarray Scanner. Fluorescent intensity data were extracted,normalized, and analyzed using Agilent Feature Extraction Software. Geneexpression was judged to be different from its reference based on anestimate of the significance of the extent of change, which wasestimated using an error model that takes into account the levels ofsignal to noise for each channel, and uses a large number of positiveand negative controls replicated on the chip to condition the estimate.Expression changes at the level of p≤0.001 were considered assignificantly different.

Statistical Considerations

The protocol called for a planned 92 patients to be enrolled of which anestimated 64 patients would be treated with therapy assigned bymolecular profiling. The other 28 patients were projected to not havemolecular profiling results available because of (a) inability to biopsythe patient; (b) no target identified by the molecular profiling; or (c)deteriorating performance status. Sixty four patients were required toreceive molecular profiling treatment in order to reject the nullhypothesis (Ho) that: ≤15% of patients would have a PFS ratio of ≥1.3(e.g. a non-promising outcome).

Treatment Selection

Treatment for the patients based on molecular profiling results wasselected using the following algorithm: 1) IHC/FISH and microarrayindicates same target; 2) IHC positive result alone; 3) microarraypositive result alone. The patient's physician was informed of suggestedtreatment and the patient was treated based on package insertrecommendations. Disease status was assessed every 8 weeks. Adverseeffects were assessed by NCI CTCAE version 3.0.

The targets and associated drugs are listed in Table 17.

TABLE 17 Pairings of Targets and Drugs Potential Target Agents Suggestedas Interacting With the Target IHC EGFR Cetuximab, erlotinib, gefitinibSPARC Nanoparticle albumin-bound paclitaxel c-KIT Imatinib, sunitinib,sorafenib ER Tamoxifen, aromatase inhibitors, toremifene, progestationalagent PR Progestational agents, tamoxifen, aromatase inhibitor,goserelin Androgen Flutamide, abarelix, bicalutamide, leuprolide,goserelin receptor PGP Avoid natural products, doxorubicin, etoposide,docetaxel, vinorelbine HER2/NEU Trastuzumab PDGFR Sunitinib, imatinib,sorafenib CD52 Alemtuzumab CD25 Denileukin diftitox HSP90 Geldanamycin,CNF2024 TOP2A Doxorubicin, epirubicin, etoposide Microarray ADAPentostatin, cytarabine AR Flutamide, abarelix, bicalutamide,leuprolide, goserelin ASNA Asparaginase BCL2 Oblimersen sodium† BRCA2Mitomycin CD33 Gemtuzumab ozogamicin CDW52 Alemtuzumab CES-2 IrinotecanDCK Gemcitabine DNMT1 Azacitidine, decitabine EGFR Cetuximab, erlotinib,gefitinib ERBB2 Trastuzumab ERCC1 Cisplatin, carboplatin, oxaliplatinESR1 Tamoxifen, aromatase inhibitors, toremifene, progestational agentFOLR2 Methotrexate, pemetrexed GART Pemetrexed GSTP1 Platinum HDAC1Vorinostat HIF1α Bevacizumab, sunitinib, sorafenib HSPCA Geldanamycin,CNF2024 IL2RA Aldesleukin KIT Imatinib, sunitinib, sorafenib MLH-1Gemcitabine, oxaliplatin MSH1 Gemcitabine MSH2 Gemcitabine, oxaliplatinNFKB2 Bortezomib NFKB1 Bortezomib OGFR Opioid growth factor PDGFCSunitinib, imatinib, sorafenib PDGFRA Sunitinib, imatinib, sorafenibPDGFRB Sunitinib, imatinib, sorafenib PGR Progestational agents,tamoxifen, aromatase inhibitors, goserelin POLA Cytarabine PTENRapamycin (if low) PTGS2 Celecoxib RAF1 Sorafenib RARA Bexarotene,all-trans-retinoic acid RXRB Bexarotene SPARC Nanoparticle albumin-boundpaclitaxel SSTR1 Octreotide TK1 Capecitabine TNF Infliximab TOP1Irinotecan, topotecan TOP2A Doxorubicin, etoposide, mitoxantrone TOP2BDoxorubicin, etoposide, mitoxantrone TXNRD1 Px12 TYMS Fluorouracil,capecitabine VDR Calcitriol VEGF Bevacizumab, sunitinib, sorafenib VHLBevacizumab, sunitinib, sorafenib ZAP70 Geldanamycin, CNF2024

Results

The distribution of the patients is diagrammed in FIG. 30 and thecharacteristics of the patients shown in Tables 18 and 19. As can beseen in FIG. 30, 106 patients were consented and evaluated. There were20 patients who did not proceed with molecular profiling for the reasonsoutlined in FIG. 30 (mainly worsening condition or withdrawing theirconsent or they did not want any additional therapy). There were 18patients who were not treated following molecular profiling (mainly dueto worsening condition or withdrawing consent because they did not wantadditional therapy). There were 68 patients treated, with 66 of themtreated according to molecular profiling results and 2 not treatedaccording to molecular profiling results. One of the two was treatedwith another agent because the clinician caring for the patient felt asense of urgency to treat and the other was treated with another agentbecause the insurance company would not cover the molecular profilingsuggested treatment.

The median time for molecular profiling results being made accessible toa clinician was 16 days from biopsy (range 8 to 30 days) and a median of8 days (range 0 to 23 days) from receipt of the tissue sample foranalysis. Some modest delays were caused by the local teams not sendingthe patients' blocks immediately (due to their need for a pathologyworkup of the specimen). Patient tumors were sent from 9 sitesthroughout the United States including: Greenville, S. C.; Tyler, Tex.;Beverly Hills, Calif.; Huntsville, Ala.; Indianapolis, Ind.; SanAntonio, Tex.; Scottsdale, Ariz. and Los Angeles, Calif.

Table 19 details the characteristics of the 66 patients who hadmolecular profiling performed on their tumors and who had treatmentaccording to the molecular profiling results. As seen in Table 19, ofthe 66 patients the majority were female, with a median age of 60 (range27-75). The number of prior treatment regimens was 2-4 in 53% ofpatients and 5-13 in 38% of patients. There were 6 patients (9%), whohad only 1 prior therapy because no approved active 2^(nd) line therapywas available. Twenty patients had progressed on prior phase Itherapies. The majority of patients had an ECOG performance status of 1.

TABLE 18 Patient Characteristics (n = 66) Characteristic n % GenderFemale 43 65 Male 23 35 Age Median (range) 60 (27-75) Number of PriorTreatments 2-4* 35 53 5-13 25 38 ECOG 0 18 27 1 48 73 *Note: 6 patients(9%) had 1 prior

As seen in Table 19, tumor types in the 66 patients included breastcancer 18 (27%), colorectal 11 (17%), ovarian 5 (8%), and 32 patients(48%) were in the miscellaneous categories. Many patients had the morerare types of cancers.

TABLE 19 Patient Tumor Types (n = 66) Tumor Type n % Breast 18 27Colorectal 11 17 Ovarian 5 8 Miscellaneous 32 48 Prostate 4 6 Lung 3 5Melanoma 2 3 Small cell (esopha/retroperit) 2 3 Cholangiocarcinoma 2 3Mesothelioma 2 3 H&N (SCC) 2 3 Pancreas 2 3 Pancreas neuroendocrine 11.5 Unknown (SCC) 1 1.5 Gastric 1 1.5 Peritoneal pseudomyxoma 1 1.5 AnalCanal (SCC) 1 1.5 Vagina (SCC) 1 1.5 Cervis 1 1.5 Renal 1 1.5 Eccrineseat adenocarinoma 1 1.5 Salivary gland adenocarinoma 1 1.5 Soft tissuesarcoma (uterine) 1 1.5 GIST (Gastric) 1 1.5 Thyroid-Anaplastic 1 1.5

Primary Endpoint: PFS Ratio ≥1.3

As far as the primary endpoint for the study is concerned (PFS ratio of≥1.3), in the 66 patients treated according to molecular profilingresults, the number of patients with PFS ratio greater or equal to 1.3was 18 out of the 66 or 27%, 95% CI 17-38% one-sided, one-sample nonparametric test p=0.007. The null hypothesis was that ≤15% of thispatient population would have a PFS ratio of ≥1.3. Therefore, the nullhypothesis is rejected and our conclusion is that this molecularprofiling approach is beneficial. FIG. 31 details the comparison of PFSon molecular profiling therapy (the bar) versus PFS (TTP) on thepatient's last prior therapy (the boxes) for the 18 patients. The medianPFS ratio is 2.9 (range 1.3-8.15).

If the primary endpoint is examined, as shown in Table 20, a PFS ratio≥1.3 was achieved in 8/18 (44%) of patients with breast cancer, 4/11(36%) patients with colorectal cancer, 1/5 (20%) of patients withovarian cancer and 5/32 (16%) patients in the miscellaneous tumor types(note that miscellaneous tumor types with PFS ratio ≥1.3 included: lung1/3, cholangiocarcinoma 1/3, mesothelioma 1/2, eccrine sweat gland tumor1/1, and GIST (gastric) 1/1).

TABLE 20 Primary Endpoint - PFS Ratio ≥1.3 By Tumor Type Total Numberwith PFS Tumor Type Treated Ratio ≥1.3 % Breast 18 8 44 Colorectal 11 436 Ovarian 5 1 20 Miscellaneous* 32 5 16 Total 66 18 27 *lung 1/3,cholangiocarcinoma ½, mesothelioma ½, eccrine sweat 1/1, GIST (gastric)1/1

The treatment that the 18 patients with the PFS ≥1.3 received based onprofiling is detailed in Table 21. As can be seen in that table forbreast cancer patients, the treatment ranged from diethylstibesterol tonab paclitaxel+gemcitabine to doxorubicin. Treatments for patients withother tumor types are also detailed in Table 21. The table further showsa comparison of the drugs that the responding patients received versusthe drugs that would have been suggested without molecular profiling andindicates which targets were used to suggest the therapies. Overall, 14were treated with combinations and 4 were treated with single agents.

TABLE 21 Targets Noted in Patients' Tumors, Treatment Suggested on theBasis of These Results, and Treatment Investigator Would Use if NoTarget Was Identified (in patients with PFS ratio ≥1.3) Treatment theTreatment Suggested Investigator Would Targets Used to Suggest on Basisof Patient's Have Used if No Results Location of Primary Treatment andMethod Tumor Molecular From Molecular Tumor Used Profiling ProfilingBreast ESR1: I; ESR1: M DES 5 mg TID Investigational CholangiocarcinomaEGFR: I; TOP1: M CPT-11 350 mg/m² every Investigational 3 weeks;cetuximab 400 mg/m² day 1, 250 mg/m² every week Breast SPARC: I; SPARC,NAB paclitaxel 260 Docetaxel, trastuzumab ERBB2: M mg/m² every 3 weeks;trastuzumab 6 mg/kg every 3 weeks Eccrine sweat gland c-KIT: I; c-KIT: MSunitinib 50 mg/d, 4 Best supportive care (right forearm) weeks on/2weeks off Ovary HER2/NEU, ER: I; Lapatinib 1,250 mg PO BevacizumabHER2/NEU: M days 1-21; tamoxifen 20 mg PO Colon/rectum PDGFR, c-KIT: II; CPT-11 70 mg/m² Cetuximab PDGFR, TOP1: M weekly for 4 weeks on/2weeks off; sorafenib 400 mg BID Breast SPARC: I; DCK: M NAB paclitaxel90 Mitomycin mg/m² every 3 weeks; gemcitabine 750 mg/m² days 1, 8, 15,every 3 weeks Breast ER: I; ER, TYMS: M Letrozole 2.5 mg daily;Capecitabine capecitabine 1,250 mg/m² BID, 2 weeks on/1 week offMalignant mesothelioma MLH1, MLH2: I; Gemcitabine 1,000 GemcitabineRRM2B, RRM1, RRM2, mg/m2 days 1 and 8, TOP2B: M every 3 weeks; etoposide50 mg/m² 3 days every 3 weeks Breast MSH2 Oxaliplatin 85 mg/m²Investigational every 2 weeks; fluorouracil (5FU) 1,200 mg/m² days 1 and2, every 2 weeks; trastuzumab 4 mg/kg day 1, 2 mg/kg every weekNon-small-cell lung EGFR: I; EGFR Cetuximab 400 mg/m² Vinorelbine cancerday 1, 250 mg/m² every week; CPT-11 125 mg/m² weekly for 4 weeks on/2weeks off Colon/rectum MGMT Temozolomide 150 Capecitabine mg/m² for 5days every 4 weeks; bevacizumab 5 mg/kg every 2 weeks Colon/rectumPDGFR, c-KIT: I; Mitomycin 10 mg once Capecitabine PDGFR: KDR, HIF1A,every 4-6 weeks; BRCA2: M sunitinib 37.5 mg/d, 4 weeks on/2 weeks offBreast DCK, DHFR: M Gemcitabine 1,000 Best supportive care mg/m² days 1and 8 every 3 weeks; pemetrexed 500 mg/m² days 1 and 8, every 3 weeksBreast TOP2A: I; TOP 2A: M Doxorubicin 50 mg/m² Vinorelbine every 3weeks Colon/rectum MGMT, VEGFA, HIF1A: Temozolomide 150 Panitumumab Mmg/m² for 5 days every 4 weeks; sorafenib 400 mg BID Breast ESR1, PR: I;ESR1, PR: Exemestane 25 mg every Doxorubicin liposomal M day GIST(stomach) EGFR: I; EGFR, RRM2: Gemcitabine 1,000 None M mg/m² days 1, 8,and 15 every 4 weeks; cetuximab 400 mg/m² day 1, 250 mg/m² every week *Abbreviations used in Table 21: I, immunohistochemistry; M, microarray;DES, diethylstilbestrol; CPT-11, irinotecan; TID, three times a day;NAB, nanoparticle albumin bound; PO, orally; BID, twice a day; GIST, GIstromal tumor.

Secondary Endpoints

The results for the secondary endpoint for this study are as follows.The frequency with which molecular profiling of a patients' tumoryielded a target in the 86 patients where molecular profiling wasattempted was 84/86 (98%). Broken down by methodology, 83/86 (97%)yielded a target by IHC/FISH and 81/86 (94%) yielding a target bymicroarray. RNA was tested for integrity by assessing the ratio of 28Sto 18S ribosomal RNA on an Agilent BioAnalyzer. 83/86 (97%) specimenshad ratios of 1 or greater and gave high intra-chip reproducibilityratios. This demonstrates that very good collection and shipment ofpatients' specimens throughout the United States and excellent technicalresults can be obtained.

By RECIST criteria in 66 patients, there was 1 complete response and 5partial responses for an overall response rate of 10% (one CR in apatient with breast cancer and PRs in breast, ovarian, colorectal andNSCL cancer patients). Patients without progression at 4 months included14 out of 66 or 21%.

In an exploratory analysis, a waterfall plot for all patients formaximum % change of the summed diameters of target lesions with respectto baseline diameters was generated. The patients who had progressionand the patients who had some shrinkage of their tumor sometime duringtheir course along with those partial responses by RECIST criteria isdemonstrated in FIG. 32. There is some shrinkage of patient's tumors inover 47% of the patients (where 2 or more evaluations were completed).

Other Analyses—Safety

As far as safety analyses there were no treatment related deaths. Therewere nine treatment related serious adverse events including anemia (2patients), neutropenia (2 patients), dehydration (1 patient),pancreatitis (1 patient), nausea (1 patient), vomiting (1 patient), andfebrile neutropenia (1 patient). Only one patient (1.5%) wasdiscontinued due to a treatment related adverse event of grade 2fatigue.

Other Analyses—Relationship Between What the Clinician Caring for thePatient would have Selected Versus What the Molecular Profiling Selected

The relationship between what the clinician selected to treat thepatient before knowing what molecular profiling results suggested fortreatment was also examined. As detailed in FIG. 33, there is no patternbetween the two. More specifically, no matches for the 18 patients withPFS ratio ≥1.3 were noted.

The overall survival for the 18 patients with a PFS ratio of ≥1.3 versusall 66 patients is shown in FIG. 34. This exploratory analysis was doneto help determine if the PFS ratio had some clinical relevance. Theoverall survival for the 18 patients with the PFS ratio of ≥1.3 is 9.7months versus 5 months for the whole population—log rank 0.026. Thisexploratory analysis indicates that the PFS ratio is correlated with theclinical parameter of survival.

Conclusions

This prospective multi-center pilot study demonstrates: (a) thefeasibility of measuring molecular targets in patients' tumors from 9different centers across the US with good quality and sufficient tumorcollection—and treat patients based on those results; (b) this molecularprofiling approach gave a longer PFS for patients on a molecularprofiling suggested regimen than on the regimen they had just progressedon for 27% of the patients (confidence interval 17-38%) p=0.007; and (c)this is a promising result demonstrating use and benefits of molecularprofiling.

The results also demonstrate that patients with refractory cancer cancommonly have simple targets (such as ER) for which therapies areavailable and can be beneficial to them. Molecular profiling forpatients who have exhausted other therapies and who are perhapscandidates for phase I or II trials could have this molecular profilingperformed.

Example 2: Molecular Profiling System

Molecular profiling is performed to determine a treatment for a disease,typically a cancer. Using a molecular profiling approach, molecularcharacteristics of the disease itself are assessed to determine acandidate treatment. Thus, this approach provides the ability to selecttreatments without regard to the anatomical origin of the diseasedtissue, or other “one-size-fits-all” approaches that do not take intoaccount personalized characteristics of a particular patient'saffliction. The profiling comprises determining gene and gene productexpression levels, gene copy number and mutation analysis. Treatmentsare identified that are indicated to be effective against diseased cellsthat overexpress certain genes or gene products, underexpress certaingenes or gene products, carry certain chromosomal aberrations ormutations in certain genes, or any other measureable cellularalterations as compared to non-diseased cells. Because molecularprofiling is not limited to choosing amongst therapeutics intended totreat specific diseases, the system has the power to take advantage ofany useful technique to measure any biological characteristic that canbe linked to a therapeutic efficacy. The end result allows caregivers toexpand the range of therapies available to treat patients, therebyproviding the potential for longer life span and/or quality of life thantraditional “one-size-fits-all” approaches to selecting treatmentregimens.

FIG. 35 illustrates a molecular profiling system that performs analysisof a cancer sample using a variety of components that measure expressionlevels, chromosomal aberrations and mutations. The molecular “blueprint”of the cancer is used to generate a prioritized ranking of druggabletargets and/or drug associated targets in tumor and their associatedtherapies.

A system for carrying out molecular profiling according to the inventioncomprises the components used to perform molecular profiling on apatient sample, identify potentially beneficial and non-beneficialtreatment options based on the molecular profiling, and return a reportcomprising the results of the analysis to the treating physician orother appropriate caregiver.

Formalin-fixed paraffin-embedded (FFPE) are reviewed by a pathologistfor quality control before subsequent analysis. Nucleic acids (DNA andRNA) are extracted from FFPE tissues after microdissection of the fixedslides. Nucleic acids are extracted using phenol-chlorform extraction ora kit such as the QIAamp DNA FFPE Tissue kit according to themanufacturer's instructions (QIAGEN Inc., Valencia, Calif.).

Gene expression analysis is performed using an expression microarray orqPCR (RT-PCR). The qPCR can be performed using a low density microarray.In addition to gene expression analysis, the system can perform a set ofimmunohistochemistry assays on the input sample. Gene copy number isdetermined for a number of genes via FISH (fluorescence in situhybridization) and mutation analysis is done by DNA sequencing(including sequence sensitive PCR assays and fragment analysis such asRFLP, as desired) for a several specific mutations. All of this data isstored for each patient case. Data is reported from the expression, IHC,FISH and DNA sequencing analysis. All laboratory experiments areperformed according to Standard Operating Procedures (SOPs).

Expression can be measured using real-time PCR (qPCR, RT-PCR). Theanalysis can employ a low density microarray. The low density microarraycan be a PCR-based microarray, such as a Taqman™ Low Density Microarray(Applied Biosystems, Foster City, Calif.).

Expression can be measured using a microarray. The expression microarraycan be an Agilent 44K chip (Agilent Technologies, Inc., Santa Clara,Calif.). This system is capable of determining the relative expressionlevel of roughly 44,000 different sequences through RT-PCR from RNAextracted from fresh frozen tissue. Alternately, the system uses theIllumina Whole Genome DASL assay (Illumina Inc., San Diego, Calif.),which offers a method to simultaneously profile over 24,000 transcriptsfrom minimal RNA input, from both fresh frozen (FF) and formalin-fixedparaffin embedded (FFPE) tissue sources, in a high throughput fashion.The analysis makes use of the Whole-Genome DASL Assay with UDG(Illumina, cat #DA-903-1024/DA-903-1096), the Illumina HybridizationOven, and the Illumina iScan System according to the manufacturer'sprotocols. FIG. 36 shows results obtained from microarray profiling ofan FFPE sample. Total RNA was extracted from tumor tissue and wasconverted to cDNA. The cDNA sample was then subjected to a whole genome(24K) microarray analysis using the Illumina Whole Genome DASL process.The expression of a subset of 80 genes was then compared to a tissuespecific normal control and the relative expression ratios of these 80target genes indicated in the figure was determined as well as thestatistical significance of the differential expression.

Polymerase chain reaction (PCR) amplification is performed using the ABIVeriti Thermal Cycler (Applied Biosystems, cat #9902). PCR is performedusing the Platinum Taq Polymerase High Fidelity Kit (Invitrogen, cat#11304-029). Amplified products can be purified prior to furtheranalysis with Sanger sequencing, pyrosequencing or the like.Purification is performed using CleanSEQ reagent, (Beckman Coulter, cat#000121), AMPure XP reagent (Beckman Coulter, cat #A63881) or similar.Sequencing of amplified DNA is performed using Applied Biosystem's ABIPrism 3730×1 DNA Analyzer and BigDye® Terminator V1.1 chemistry (LifeTechnologies Corporation, Carlsbad, Calif.). The BRAF V600E mutation isassessed using the FDA approved Cobas® 4800 BRAF V600 Mutation Test fromRoche Molecular Diagnostics (Roche Diagnostics, Indianapolis, Ind.).NextGeneration sequencing is performed using the MiSeq platform fromIllumina Corporation (San Diego, Calif., USA) according to themanufacturer's recommended protocols.

For RFLP, ALK fragment analysis is performed on reverse transcribed mRNAisolated from a formalin-fixed paraffin-embedded tumor sample usingFAM-linked primers designed to flank and amplify EML4-ALK fusionproducts. The assay is designed to detect variants v1, v2, v3a, v3b, 4,5a, 5b, 6, 7, 8a and 8b. Other rare translocations may be detected bythis assay; however, detection is dependent on the specificrearrangement. This test does not detect ALK fusions to genes other thanEML4.

IHC is performed according to standard protocols. IHC detection systemsvary by marker and include Dako's Autostainer Plus (Dako North America,Inc., Carpinteria, Calif.), Ventana Medical Systems Benchmark® XT(Ventana Medical Systems, Tucson, Ariz.), and the Leica/VisionBiosystems Bond System (Leica Microsystems Inc., Bannockburn, Ill.). Allsystems are operated according to the manufacturers' instructions.

FISH is performed on formalin-fixed paraffin-embedded (FFPE) tissue.FFPE tissue slides for FISH must be Hematoxylin and Eosion (H & E)stained and given to a pathologist for evaluation. Pathologists willmark areas of tumor to be FISHed for analysis. The pathologist reportmust show tumor is present and sufficient enough to perform a completeanalysis. FISH is performed using the Abbott Molecular VP2000 accordingto the manufacturer's instructions (Abbott Laboratories, Des Plaines,Iowa). ALK is assessed using the Vysis ALK Break Apart FISH Probe Kitfrom Abbott Molecular, Inc. (Des Plaines, Ill.). HER2 is assessed usingthe INFORM HER2 Dual ISH DNA Probe Cocktail kit from Ventana MedicalSystems, Inc. (Tucson, Ariz.) and/or SPoT-Light® HER2 CISH Kit availablefrom Life Technologies (Carlsbad, Calif.).

DNA for mutation analysis is extracted from formalin-fixedparaffin-embedded (FFPE) tissues after macrodissection of the fixedslides in an area that % tumor nuclei ≥10% as determined by apathologist. Extracted DNA is only used for mutation analysis if % tumornuclei ≥10%. DNA is extracted using the QIAamp DNA FFPE Tissue kitaccording to the manufacturer's instructions (QIAGEN Inc., Valencia,Calif.). DNA can also be extracted using the QuickExtract™ FFPE DNAExtraction Kit according to the manufacturer's instructions (EpicentreBiotechnologies, Madison, Wis.). The BRAF Mutector I BRAF Kit (TrimGen,cat #MH1001-04) is used to detect BRAF mutations (TrimGen Corporation,Sparks, Md.). Roche's Cobas PCR kit can be used to assess the BRAF V600Emutation. The DxS KRAS Mutation Test Kit (DxS, #KR-03) is used to detectKRAS mutations (QIAGEN Inc., Valencia, Calif.). BRAF and KRAS sequencingof amplified DNA is performed using Applied Biosystems' BigDye®Terminator V1.1 chemistry (Life Technologies Corporation, Carlsbad,Calif.).

Next generation sequencing is performed using aTruSeq/MiSeq/HiSeq/NexSeq system offered by Illumina Corporation (SanDiego, Calif.) or an Ion Torrent system from Life Technologies(Carlsbad, Calif., a division of Thermo Fisher Scientific Inc.)according to the manufacturer's instructions.

Example 3: Molecular Profiling Reports

An exemplary report generated by the molecular profiling systems andmethods of the invention is shown in FIGS. 27A-V. The figures illustratean exemplary patient report based on molecular profiling the tumor of anindividual having triple negative breast cancer. Note that the molecularprofiling results indicate ER/PR/HER2 negative on pages 3-4 (i.e., FIGS.27C-D). FIG. 27A illustrates a cover page of a report indicating patientand specimen information for the patient. FIG. 27A also displays asummary of agents associated with potential benefit and potential lackof benefit. Agents associated with potential benefit are highlighted inbold if on NCCN Compendium™ (i.e., recommended by NCCN guidelines forthe particular tumor lineage) or in plain text off NCCN Compendium™(i.e., not part of the NCCN guidelines for the particular tumorlineage). FIG. 27A also lists clinical trials which may be availablegiven the molecular profiling results, here no trials were matched. FIG.27B continues from FIG. 27A and lists agents with indeterminate benefit,indicating that the molecular profiling results were deemed notdefinitive for potential benefit and potential lack of benefit for theindicated agent. FIGS. 27C-D provide a summary of biomarker results fromthe indicated assays. FIG. 27E provides more detailed information forbiomarker profiling used to associate agents with potential benefit,whereas FIGS. 27F-G illustrate more detailed information for biomarkerprofiling used to associate agents with lack of potential benefit. FIG.27H illustrates more detailed information for biomarker profiling usedto associate agents with indeterminate benefit. FIG. 27I illustratesmore detailed information for biomarker profiling matched to potentialclinical trials. FIG. 27J, FIG. 27K, FIG. 27L, FIG. 27M and FIG. 27Nprovide a listing of published references used to provide evidence ofthe biomarker—agent association rules used to construct the report. FIG.27O presents a description of the specimen/s received and a disclaimer,e.g., that ultimate treatment decisions reside solely within thediscretion of the treating physician. FIG. 27P and FIG. 27Q provide moreinformation about the mutational analysis such as point mutationsperformed by Next Generation sequencing. FIG. 27R provides moreinformation about gene copy number variations detected by NextGeneration Sequencing and FIG. 27S provides more information about genefusions and transcript variants detect by NGS analysis of RNAtranscripts. FIG. 27T provides more information about the IHC analysisperformed on the patient sample, e.g., the staining threshold andresults for each marker. FIG. 27U provides more information about theISH analysis performed on the patient sample, which comprised CISH forthis tumor. FIG. 27V provides the framework used for the literaturelevel of evidence as included in the report.

Example 4: Molecular Profiling Service

FIGS. 26A-D illustrate a molecular profiling service requisition using amolecular profiling approach as outlined in Tables 7-9 and 12-15, andaccompanying text herein. Such requisition presents choices formolecular profiling that can be presented to a caregiver, e.g., amedical oncologist who may prescribe a therapeutic regimen to a cancerpatient. FIG. 26A shows a choice of MI Profile panel that is assessedusing multiple technologies, e.g., according to Tables 7-9, or and MIProfile X, e.g., according to Tables 7-9 with the expanded set of geneanalysis presented in Tables 12-15. Alternately, individual biomarkerscan be assessed, as shown in FIG. 26B. The individual markers mayinclude those in addition to the marker panels listed in Tables 7-9 and12-15. For example, H3K36me3, PBRM1 and PD-1 may be made available. FIG.26C amd FIG. 26D illustrate sample requirements that can be used toperform molecular profiling on a patient tumor sample according to thebiomarker choices in FIGS. 26A-B. FIG. 26C provides requirements forformalin fixed paraffin embedded (FFPE) and FIG. 26D providesrequirements for fresh samples. In the event that insufficient quantityor tissue, bodily fluid or percent tumor is available to perform alltests desired to be performed, certain tests can be prioritized, e.g.,according to physician preference or experience with the variousbiomarkers in similar tumor types.

Example 5: Biomarker-Drug Associations

Molecular profiling according to the invention leverages multipletechnologies to provide evidence-based, clinically actionableinformation FDA approved cancer drugs. This Example summarizes exemplarybiomarker—drug associations available with Level 1 or Level 2 evidence.As described above, Level 1 evidence comprises very high level ofevidence. For example, the treatment comprises the standard of care.Level 2 evidence comprises high level of evidence but perhapsinsufficient to be considered for standard of care. Table 22 lists 32drugs whose biomarker—drug associations are based on IHC or IHC/ISHcombination. Table 23 lists 9 drugs whose biomarker—drug associationsare based on sequencing/IHC combination. Table 24 lists 7 drugs whosebiomarker—drug associations are based on sequencing alone. Thesequencing can comprise, e.g., Next Generation Sequencing (NGS), Sangersequencing, qPCR, or any combination thereof.

For each row in Tables 22-24, the markers and technologies are listed inrespective order. For example, in the fourth row in Table 22, drug name“ado-trastuzumab emtansine (T-DM1)”, the markers “Her2/Neu, Her2/Neu”are assessed by “FISH” and “IHC,” respectively. As another example, inthe eighth row in Table 22, drug name “crizotinib”, the markers “ALK,ROS1” are assessed by “FISH” and “FISH,” respectively.

TABLE 22 Drugs Associations supported by Evidence by IHC and ISHIllustrative Drug Name Markers Technologies abarelix Androgen ReceptorIHC abiraterone Androgen Receptor IHC ado-trastuzumab emtansine (T-Her2/Neu, Her2/Neu FISH, IHC DM1) anastrozole ER, PR IHC, IHCbicalutamide Androgen Receptor IHC capecitabine TS IHC crizotinib ALK,ROS1 FISH, FISH degarelix Androgen Receptor IHC docetaxel SPARCPolyclonal, TUBB3, SPARC IHC, IHC, IHC, IHC, Monoclonal, PGP, TLE3 IHCdoxorubicin TOP2A, TOP2A, Her2/Neu, PGP FISH, IHC, FISH, IHCenzalutamide Androgen Receptor IHC epirubicin TOP2A, PGP, TOP2A,Her2/Neu FISH, IHC, IHC, FISH exemestane ER, PR IHC, IHC fluorouracil TSIHC flutamide Androgen Receptor IHC fulvestrant ER, PR IHC, IHCgemcitabine RRM1 IHC goserelin PR, ER, AR IHC, IHC, IHC irinotecan TOPO1IHC lapatinib Her2/Neu, Her2/Neu FISH, IHC letrozole ER, PR IHC, IHCleuprolide ER, PR IHC, IHC liposomal-doxorubicin TOP2A, TOP2A, PGP,Her2/Neu FISH, IHC, IHC, FISH megestrol acetate PR, ER IHC, IHCnab-paclitaxel SPARC Monoclonal, SPARC Polyclonal, TLE3, IHC, IHC, IHC,IHC, PGP, TUBB3 IHC paclitaxel TUBB3, SPARC Polyclonal, TLE3, SPARC IHC,IHC, IHC, IHC, Monoclonal, PGP IHC pemetrexed TS IHC pertuzumabHer2/Neu, Her2/Neu IHC, FISH tamoxifen PR, ER IHC, IHC topotecan TOPO1IHC toremifene PR, ER IHC, IHC triptorelin Androgen Receptor IHC

TABLE 23 Drugs Associations supported by evidence by IHC, ISH andSequencing Drug Name Markers Illustrative Technologies cetuximab PTEN,EGFR, BRAF, KRAS, IHC, IHC, Sanger SEQ/NGS, Sanger NRAS, PIK3CA SEQ/NGS,Sanger SEQ/NGS, Sanger SEQ/NGS dacarbazine MGMT, MGMT, MGMT, IDH1 MGMTMethylation, Pyro SEQ, IHC, NGS erlotinib PTEN, KRAS, cMET, PIK3CA, IHC,Sanger SEQ/NGS, FISH, Sanger EGFR SEQ/NGS, Sanger SEQ/NGS everolimusHer2/Neu, PIK3CA, Her2/Neu, IHC, Sanger SEQ/NGS, Sanger SEQ/NGS, ERFISH, Sanger SEQ/NGS, IHC, IHC gefitinib PTEN, EGFR, PIK3CA, cMET, IHC,Sanger SEQ/NGS, Sanger SEQ/NGS, KRAS, cMET IHC, Sanger SEQ/NGS, FISHpanitumumab KRAS, BRAF, NRAS, PTEN, Sanger SEQ/NGS, Sanger SEQ/NGS,Sanger PIK3CA SEQ/NGS, IHC, Sanger SEQ/NGS temozolomide MGMT, MGMT,IDH1, MGMT MGMT Methylation, Pyro SEQ, NGS, IHC temsirolimus PIK3CA,Her2/Neu, Her2/Neu, Sanger SEQ/NGS, Sanger SEQ/NGS, Sanger ER SEQ/NGS,FISH, IHC, IHC, IHC trastuzumab Her2/Neu, PTEN, PIK3CA, FISH, IHC,Sanger SEQ/NGS, IHC Her2/Neu

TABLE 24 Drugs Associations supported by evidence by Sequencing DrugName Markers Illustrative Technologies afatinib EGFR Sanger SEQ/NGSdabrafenib BRAF, BRAF Sanger SEQ/NGS, qPCR imatinib c-KIT, PDGFRA NGS,NGS sunitinib c-KIT NGS trametinib BRAF, BRAF Sanger SEQ/NGS, qPCRvandetanib RET NGS vemurafenib BRAF, BRAF Sanger SEQ/NGS, qPCR

Biomarker—drug associations can be updated as additional informationbecomes available. For example, new literature reports, clinical triallistings or clinical trial data may provide new and/or updatedbiomarker—drug associations or clinical trial associations. Theinvention may also rely upon previous molecular profiling results toupdate biomarker—drug associations. For example, comparison of molecularprofiling results against actual treatments and outcomes may suggestupdated biomarker—drug associations where the status of certainbiomarkers correlates with benefit or lack of benefit for certain drugs.

Example 6: Molecular Profiling Reagents

Molecular profiling according to the invention is performed usingvarious analysis methods as described herein. The analysis includessequence variant analysis (e.g., Sanger sequencing, Next GenerationSequencing (NGS) or pyrosequencing), immunohistochemistry (proteinexpression), CISH or FISH (gene amplification), and/or RNA fragmentanalysis (FA). Various reagents used for IHC and ISH analysis asdescribed herein are shown in Table 25.

TABLE 25 Reagents used for molecular profiling Product Name VendorCatalog Number Clone AR antibody LEICA NCL-AR-318 AR27 Chr7 CISH probeVENTANA 760-1219 (PROBE) cMet antibody VENTANA 790-4430 SP44 cMet CISHprobe VENTANA 760-1228 (PROBE) EGFR antibody DAKO K1494 2-18C9 ERantibody VENTANA 790-4325 SP1 ERCC1 antibody ABCAM AB2356 8F1 HER2 CISHprobe VENTANA 780-4422 (PROBE) HER2/neu antibody VENTANA 790-2991 4B5MGMT antibody INVITROGEN 18-7337 MT23.2 NEGATIVE MOUSE VENTANA 760-2014MOPC21 NEGATIVE MOUSE DAKO IR750 NEGATIVE RABBIT VENTANA 760-1029 (POLY)NEGATIVE RABBIT DAKO IR600 PGP (MDR1) antibody INVITROGEN 18-7243 C494PR antibody VENTANA 790-4296 IE2 PTEN antibody DAKO M 3627 6H2.1 RRM1antibody PROTEINTECH 10526-1-AP (POLY) SPARC-MONO antibody R&D SYSTEMSMAB941 122511 SPARC-POLY antibody EXALPHA X1867P (POLY) TLE3 antibodySANTA CRUZ SC-9124 (POLY) TOPO2A antibody LEICA NCL-TOPO11A 3F6 TOPO1antibody LEICA NCL-TOPO1 1D6 TS antibody INVITROGEN 18-0405 TS106/4H4B1TUBB3 antibody COVANCE PRB-435P (POLY) MLH-1 antibody VENTANA 790-4535M1 MSH-2 antibody VENTANA (CELL 760-4265 G219-1129 MARQUE) MSH-6antibody VENTANA 790-4455 44 PMS-2 antibody VENTANA (CELL 760-4531EPR3947 MARQUE) PD-1 antibody BD PHARMINGEN 562138 EH12.1 PD-L1 antibodyR&D SYSTEMS MAB1561 130021 PBRM1 (PB1/ BETHYL A301-591A (POLY) BAF180)antibody LABORATORIES BAP1 antibody SANTA CRUZ SC-28383 C-4 SETD2(ANTI-HISTONE ABCAM AB9050 (POLY) H3) antibody

The reagents may be updated as improvements become available.

Example 7: Molecular Profiling of Immune Checkpoint Related Genes

Clinical response to immune checkpoint inhibitor therapy ranges from 18%to 28% by tumor type. There is unmet clinical need for laboratory teststhat can identify patients likely to respond to such therapy. Reportsindicate that 36% of transgenic tumors with PD-1 expression responded toanti-PD1 therapy while no PD-1 negative cases responded. Estimatedobjective responses for tumors expressing FoxP3 and IDO by IHC were10.38 and 8.72 respectively. This Example used microarray expressiondata to characterize the presence of immune response modulators in humantumors and possibly identify a subset of cases as the candidates forimmune checkpoint inhibitor therapy.

A retrospective analysis of gene expression microarray data for immunerelated genes was performed on 9,025 qualifying paraffin embedded humantumor specimens (HumanHT-12 v4 beadChip Illumina Inc., San Diego,Calif.). Samples from LN metastases were excluded from analysis. Immunecheckpoint-related genes examined included CTLA4, its binding partnersCD80 and CD86, PD-L1, CD276 (B7-H3), Granzymes A and B, CD8a, CD19 andthe chemokine receptor CCR7. The normalized expression values for thesegenes were plotted by tumor types to compare relative expression levelsand Principal Component Analysis was performed.

The results of this analysis showed that PD-L1 expression was above the90th percentile of normal control tissue in 4% of breast cancers, 3% ofrenal cancers, 7% of NSCLC, 3% ovarian cancer and 5% of colon cancertumors. Principal component analysis of the immune checkpoint-relatedgenes showed the greatest percentage of “distinct” cases within ovarian,melanoma, colon, gastric and pancreatic cancers.

Microarray analysis can identify tumors with unique immune componentsthat are more likely to respond to immune checkpoint therapy.

Example 8: Genomic and Protein Alterations in Triple Negative (TN)Metaplastic Breast Cancer

Metaplastic breast cancer (“MpBC”) is a rare subtype (less than 1% ofall breast cancers), is generally ER, PR and HER2-negative (triplenegative, “TN”), demonstrates a claudin-low gene expression profile, andis poorly responsive to cytotoxic therapy. Little is known about thegenomic alterations (GA) in MpBC nor about overexpressed proteins thatmay be amenable to targeted therapy.

Molecular profiling according as described herein was used to assess 126cases of TN MpBCs. Specific testing was performed per physician requestand included sequencing (Sanger or next generation sequencing [NGS]),protein expression (immunohistochemistry [IHC]), and/or geneamplification (CISH or FISH) as described herein.

The 126 member patient cohort had a median age of 60 years old, range21-94 (6 patients <50 years old). 81% of patients had documentedmetastatic disease. Sites of metastasis included 12 in the chestwall/skin/soft tissue, eight in the lung, four in the lymph nodes, onein the bone, and 61 unreported. By ICD-O code, 55 patients hadmetaplastic carcinoma, NOS, 23 patients had an adenocarcinoma withspindle cell metaplasia, 20 had an adenocarcinoma with squamous featuresand 8 had an adenocarcinoma with cartilage elements.

Table 26 shows the percentage of gene mutations, amplifications, and IHCfindings for biomarkers that were different between TNBC and MpBCs, as apercentage of total patients tested.

TABLE 26 Molecular Profiling differences between TNBC and MpBCs ISH, %IHC % Positive Gene Mutation, % Positive PTEN TP53 PIK3CA HRAS cMET EGFRloss AR cMET Ki67 TOPO1 TNBC 64 13 0 0 22 66 17 13 85 70 Metaplastic 3239 21 4 17 44 8 3 95 49 P value 0.101 0.002 0.002 0.430 0.801 0.0010.046 0.250 0.650 0.147

The above analysis revealed that the biomarker profile of MpBC was moresimilar to non-TNBC than to TNBC (data not shown). mTOR pathwayinvolvement (PIK3CA mutated and PTEN loss) was significantly differentbetween TNBC and MpBC. In the MpBC cohort, 2 of 14 cases had PIK3CA andTP53 co-mutated (14%), whereas in the TNBC cohort, 26 of 55 cases hadPIK3CA and TP53 co-mutated (47%).

Table 27 shows the results of IHC profiling of the MpBCs in more detail.In the table, a “$” symbol next to the biomarker name indicates thatexpression of the biomarker below the threshold is considered predictiveof response to therapy. In all other cases, expression above thethreshold is considered predictive of response to therapy. Thresholdsare set for each biomarker based on staining intensity and/or percentageof positive cells.

TABLE 27 IHC profiling of MpBCs Total Positive Total Cases Evaluated %Positive AR 8 97 8.2 BCRP 7 11 63.6 cKit 5 57 8.8 cMET 1 37 2.7 EGFR 7 977.8 ER 0 98 0 ERCC1 19 40 47.5 HER2 0 99 0 MGMT^($) 39 69 56.5 MRP1 4654 85.2 p53 20 42 48.6 PDGFR 5 22 22.7 PGP 8 82 9.8 PR 2 98 2 PTEN^($)55 100 55 RRM1^($) 20 63 31.7 SPARC 40 92 43.5 TLE3 32 87 36.8 TOP2A 3758 63.8 TOPO1 28 56 50 TS^($) 42 81 51.9 TUBB3^($) 17 25 68

FIG. 37A shows selected results of mutational analysis deteted by Sangersequencing or NGS along with suggested therapy. Mutations were notdetected in this cohort in the following genes: ABL1, AKT1, ALK, APC,ATM, CDH1, cKIT, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2,FLT3, GNA11, GNAQ, GNAS, HNF1A, IDH1, JAK2, KDR, KRAS, MPL, NOTCH1,NPM1, NRAS, PDGFRA, RET, SMAD4, SMARCB1, SMO, STK11, VHL. A breakdown ofspecific mutations in the genes indicated in FIG. 37A is shown in Table28:

TABLE 28 Mutations observed in MpBCs Gene Mutation # Observed Exon APCL1129S 1 16 BRAF N581I 1 15 cMET T1010I 1 14 HRAS G12D 1 2 G13V 1 2 Q61L1 3 MLH1 S406N 1 12 PIK3CA E545K 1 9 G106R 1 1 H1047L 2 20 H1047R 10 20N345K 1 4 PTEN R233X 1 7 JAK3 V722I 1 16 TP53 S106R 1 4 Y163C 1 5 R213X1 6 G244S 1 7 Y236S 1 7 D281E 1 8 R273H 1 8 R333fs 1 10

FIG. 37B and Table 29 present comparison of p53 Mutated, PIK3CA Mutated,and EGFR amplified MpBC patients. Table 29 shows patient characteristicsof those harboring mutations in PIK3CA and p53/TP53, and amplificationof EGFR. FIG. 37B shows a selection of molecular alterations detected inthese tumors as indicated.

TABLE 29 Patient Characteristics PIK3CA TP53 EGFR MT, n = 14 MT, n = 8Amp, n = 4 Median Age 65 51 63 Percent 85.7% 87.5% 75.0% MetastaticHistology, Sarcomatoid = 2 Sarcomatoid = 0 Sarcomatoid = 2 MetaplasticSquamous = 6 Squamous = 2 Squamous = 1 NOS = 5 NOS = 6 NOS = 1

FIGS. 37C-D present further comparison of PIK3CA mutant vs. TP53 mutantvs. EGFR amplified MpBC for individual patients. FIG. 37C presents datafor fourteen PIK3CA mutant patients. Under “Demographics,” “Met?”indicates whether the cancer is metastatic. Under the different analysistechniques (i.e., IHC, ISH and DNA Sequencing), the biomarker isindicated, “n/t” means non-tested, and a check mark indicates anactionable finding (i.e., suggesting potential drug therapy). UnderKi67%, the percentage Ki67 positivity is indicated and under PIK3CA, thespecific mutation is indicated. FIG. 37D is similar to FIG. 37C exceptthat data for TP53 mutant MpBCs and EGFR amplified MpBCs is shown. Thesedata suggest that subgroups within MpBC may have different pathways oforigin and therapy oppportunities. For example, EGFR Amplified MpBCs mayhave lower incidence of MGMT underexpression but higher incidence ofSPARC expression as compared to PIK3CA and TP53 mutants.

FIG. 37E presents further Ki67 analysis, a proliferative marker, for 64patients. The median Ki67 percent positive cells was 46.7%.Proliferation of MpBC is highly variable, reflective of the indolent tohighly proliferative spectrum of progression seen in MpBC, compared toTNBC, which tends to be more proliferative. Six cases were both ARpositive/Ki67>20% (median Ki67 for AR+MpBC=24).

FIG. 37F indicates potential therapeutic strategies suggested bymolecular evaluation of MpBC by IHC (immunohistochemistry) and/or ISH(in situ hybridization). The figure shows results for a selection ofbiomarkers assessed by IHC and EGFR by ISH (bar labeled “EGFR amp”). Thex-axis indicates the biomarker and whether it was detected as high orlow (depending on actionability) in the indicated number of patients.Potential targeted drug therapies are shown above each bar. The Ki67spectrum reflects variable history and spread between indolent andaggressive progression. PD-1 (71%) and PD-L1 (100%) were expressed athigh levels in the samples tested.

Comparison of the genomic and protein expression profiles highlightssome differences between the two cancers. Although poorly responsive tocytotoxic therapies, molecular alterations identified in 97% of cases inthis large series by multiplatform profiling points to many potentialtherapeutic strategies for MpBCs, including: mTOR pathways inhibitorssuggested by gene alterations in the PI3K pathway (52% of cases hadPTEN/PIK3CA mutations or PTEN loss); immunomodulatory agents, approvedor currently in clinical trials, suggested by the presence ofPD-1/PD-L1; gemcitabine treatment suggested by low RRM1 expression in68% of MpBCs; imitinab or anti-androgen therapies suggested by cKIT (9%)and AR protein overexpression (8%); MEK inhibitors suggested by HRASmutations (21%) and BRAF mutations (2%). Other potential therapeuticallytargetable gene alterations were present at low incidence, thusindicating a benefit of comprehensive molecular profiling in thesepatients. These results highlight the benefit of comprehensive molecularprofiling of the invention to identify both common and potentially raretumor characteristics that can guide therapeutic strategy.

REFERENCES

-   1. Song, Y, et al. Unique clinicopathological features of    metaplastic breast carcinoma compared with invasive ductal carcinoma    and poor prognostic indicators. World Journal of Surgical Oncology    2013, 11:129.-   2. Cooper et al. Molecular alterations in metaplastic breast    carcinoma. J Clin Pathol. 2013 June; 66(6):522-8.-   3. Hu et al. Current progress in the treatment of metaplastic breast    carcinoma. Asian Pac J Cancer Prev.

2013; 14(11):6221-5.

Example 9: PD1 and PDL1 in HPV+ and HPV−/TP53 Mutated Head and NeckSquamous Cell Carcinomas

This Example investigated the role of the programmed death 1 (PD1) andprogrammed death ligand 1 (PDL1) immunomodulatory axis in head and necksquamous cell carcinoma (HNSCC), a cancer with viral and non-viraletiologies. Determination of the impact of this testing in humanpapilloma virus (HPV)-positive and HPV-negative/TP53-mutated HNSCCcarries great importance due to the development of new immunomodulatoryagents.

Thirty-four HNSCC cases, including 16 HPV+ and 18 HPV−/TP53 mutant, wereanalyzed for the PD1/PDL1 immunomodulatory axis by immunohistochemicalmethods. HNSCC arising in the following anatomic sites were assessed:pharynx, larynx, mouth, parotid gland, paranasal sinuses, tongue andmetastatic SCC consistent with head and neck primary.

Results are summarized in FIG. 38. 8/34 (24%) HNSCC were positive forcancer cells expression of PDL1, and 13/34 (38%) HNSCC were positive forPD1+ tumor infiltrating lymphocytes (TILs). 3/34 (8.8%) were positivefor both components of the PD1/PDL1 axis. Comparison of PD1 and PDL1expression in HPV+ and HPV−/TP53mutant HNSCC showed PD1+ TILs were morefrequent in HPV+vs. HPV-HNSCC (56% vs. 22%; p=0.07), whereas PDL1+ tumorcells more frequent in HPV− vs. HPV+HNSCC (38% vs. 13%; p=0.14). PD1 andPDL1 were expressed in both oropharyngeal and non-oropharyngeal HNSCC:33% vs. 39% for PD1+ TILs, respectively, and 11% and 33% for PDL-1,respectively. To examine the role of PD1 and PDL1 in progression ofdisease, expression was compared between metastatic and non-metastaticHNSCC. PD1+ TILs were detected in 45% of metastatic vs. 25%non-metastatic HNSCC (p=0.29), and PDL1 was detected in 27% vs. 17% ofmetastatic vs. non-metastatic HNSCC. Interestingly, the three cases thatwere positive for both PD1 and PDL1 were metastatic HNSCC, including atumor of the mandible which had metastasized to the bone of the arm, andtwo unknown primary consistent with head and neck primary, onemetastatasized to the lymph nodes and the other metastasized to thelung.

Immune evasion through the PD1/PDL1 axis is relevant to both viral (HPV)and non-viral (TP53) etiologies of HNSCC. Expression of both axiscomponents was less frequently observed across HNSCC tumor sites, andelevated expression of both PD1 and PLD1 was seen at a higher frequencyin metastatic HNSCC. In summary, we observed that: 1) PDL1+ TILs weremore frequent (56%) in HPV+HNSCC; 2) PD1 expression was more frequent(38%) in HPV−/TP53 mutated HNSCC; 3) elevation of both components of theaxis (PD1 and PDL1), occurs at low frequency (8%); 4) expression of PDL1and PD1 occurs in head and neck cancers that occur in oropharyngeal andnon-oropharyngeal sites; and 5) the PD1/PDL1 pathway is more frequentlyexpressed in metastatic cases vs. non-metastatic HNSCC.

Example 10: Programmed Cell Death 1 (PD-1) and its Ligand (PD-L1) inCommon Cancers and their Correlation with Molecular Cancer Type

Programmed death-1 (PD-1, CD279) is an immune suppressive molecule thatis upregulated on activated T cells and other immune cells. It isactivated by binding to its ligand PD-L1 (B7-H1, CD274), which resultsin intracellular responses that reduce T-cell activation. Aberrant PD-L1expression had been observed on cancer cells, leading to the developmentof PD-1/PD-L1-directed cancer therapies, which have shown promisingresults in late phase clinical trials. Blockade of the PD-1 and PD-L1interaction led to good clinical responses in several, but not allcancer types, and the heterogeneous cellular expression of PD-1/PD-L1may underlie these selective responses (1-6).

PD-1/PD-L1 expression has been studied by various methods in differentcancer subtypes (7). Most of the published papers focused on prognosticrelevance of PD-1/PD-L1 and less is known about their predictive valueas well as their relationship to molecular genetic alterations in solidtumors (1). In this Example, we analyzed distribution of PD-1+tumor-infiltrating lymphocytes (TIL) and PD-L1 expression in the mostcommon solid cancers and further correlated these biomarkers withgenotypic and phenotypic characteristics of tumors.

Material and Methods

Tumor Samples

The study cohort consisted of 437 tumor samples (both primary andmetastatic) representing both major and some rare solid cancer types:380 carcinomas (breast, colon, lung, pancreas, prostate, Merkel cell,ovary, liver, endometrial, bladder, kidney and cancers of unknownprimary [CUP]), 33 soft tissue sarcomas (liposarcomas, chondrosarcomas,extraskeletal myxoid chondrosarcomas and uterine sarcomas) and 24malignant cutaneous melanomas.

Molecular Methods

Tumor samples were evaluated using a commercial multiplatform approachconsisting of protein analysis (immunohistochemistry), gene copy numberanalysis (in-situ hybridization) and gene sequencing (Next-GenerationSequencing with the Illumina MiSeq platform) as described herein. Seealso reference 8.

The presence of PD-1+ lymphocytes was evaluated with monoclonal antibodyNAT105 (Cell Marque) while the expression of PD-L1 was analyzed withB7-H1 antibody (R&D Systems), using automated immunohistochemicalmethods.

Due to the biopsy size-related dependence on the detection of PD-1 TILs(9, 10), we evaluated their density using a hot-spot approach, analogousto the previously described method for measuring neoangiogenesis (11).The whole tumor sample was reviewed at a low power (4× objective) andthe area of highest density of TILs in direct contact with malignantcells of the tumor at 400× visual field (40× objective×10× ocular) wasenumerated (number of PD-1+ TIL/high power fields (hpf)). The intensityof the cancer cells expression of PD-L1 was recorded on asemiquantitative scale (0-3+): 0 for no staining, 1+ for weakcytoplasmic staining, 2+ moderate membranous and cytoplasmic stainingand 3+ strong membranous and cytoplasmic staining. Percent of tumorcells expressing PD-L1 at the highest intensity was recorded.

Statistical Methods

The 2-tail Fisher's exact test and Chi-square test were applied for thecorrelation between the variables (p≤0.05).

Results

PD-1 and PD-L1 Expression in Solid Tumors

PD-1 and PD-L1 expression in solid tumors and their subtypes aresummarized in Tables 30-33.

TABLE 30 Overview over PD-1 and PD-L1 expression in various types ofsolid tumors PD-1 PD-L1 Concurrent Tumor types expression (% (tumor PD-1and PD-L1 (n = 437 total) and range) cells) (%) expression (%)Carcinomas (n = 380 total): a) Breast (n = 116) 51% (1-20) 45% 29% b)Colon (n = 87) 50% (1->20) 21% 12% c) Non-small cell lung 75% (1-20) 50%43% cancer (n = 44) d) Pancreas (n = 23) 43% (1-16) 23%  9% e) Prostate(n = 20) 35% (1-6) 25%  5% f) Merkel cell carcinoma 17% (1-4)  0%  0% (n= 19) g) Endometrium (n = 16) 86% (1-13) 88% 79% h) Ovary (n = 14) 93%(1-16) 43% 36% i) Liver (n = 13) 38% (1-5)  8%  0% j) Bladder (n = 11)73% (1-10) 55% 55% k) Kidney (n = 11) 36% (1-3) 67% 33% l) CUP (n = 6)50% (1-4) 33% 33% Sarcomas (n = 33 total) 30% (1->10) 97% 31% Melanoma(n = 24 total) 58% (1-15) 92% 58%

TABLE 31 PD-1 and PD-L1 expression in breast cancer according to themolecular subtype PD-1 expression/ PD-L1 Concurrent Breast cancersubtypes HPF (TILs) (tumor PD-1 and PD-L1 (n = 116) (% and range) cells)(%) expression (%) Luminal tumors (n = 58) a) Luminal A (n = 33) 25%(1->10) 33% 13% b) Luminal B (n = 25) 44% (1-20) 33% 17% HER2 positive(n = 5) 60% (1-9) 20% 20% Triple-negative (n = 53) 70% (1-20)*  59%* 45%* *Significantly higher than in luminal tumors.

Table 32 shows that PD-1 and PD-L1 exhibited higher expression in tumorswith high microsatellite instability (“MSI-H”) versus microsatellitestable tumors (“MSS”). The MSI-H cases here comprised Lynch syndrome andsporadic colon cancers.

TABLE 32 PD-1 and PD-L1 expression in colorectal carcinomas inrelationship to the microsatellite instability status PD-1 expression/PD-L1 Concurrent Colon cancer subtypes HPF (TILs) (tumor PD-1/PD-L1 (n =87) (% and range) cells) (%) expression (%) MSS colon cancers 39% (1-11)13%  4% (n = 60) MSI-H colon cancers 77% (1->20)* 38%* 32%* (n = 27)*Significantly higher (p < 0.05)

TABLE 33 Overview over PD-1 and PD-L1 expression in sarcoma subtypesPD-1 expression/ PD-L1 Concurrent Sarcoma subtypes HPF (TILs) (tumorPD-1 and PD-L1 (n = 33) (% and range) cells) (%) expression (%)Liposarcoma (n = 20) 45% (1->10) 100% 45% Chondrosarcoma (n = 8) 12%(1-) 100% 12% Extraskeletal myxoid 0%  67%  0% chondrosarcoma (n = 3)Uterine sarcoma (n =2) 0% 100%  0%

PD-1+ lymphocytes were consistently identified in reactive, peri-tumorallymphoid follicles which served as an internal positive control.

PD-1+ TILs in direct contact with cancer cells were uncommon in somecancer types (e.g. 0% observed in extraskeletal myxoid chondrosarcoma inthis cohort), although triple-negative breast cancer (TNBC), bladdercancer, microsatellite instability high (MSI-H) colon cancer, non-smallcell lung cancer (NSCLC), endometrial and ovarian cancer were frequently(70-100%) infiltrated with PD-1+ TILs. When present, PD-1+ TILs densityvaried from 1 to >20/hpf. See Table 30.

PD-L1 was consistently expressed in the tumor microenvironment includingendothelial cells, macrophages and dendritic cells, at strong (2+/3+)intensity and was used as internal positive control. In contrast, thecancer cells expressed PD-L1 at widely varying levels and proportions.Consistent, strong membranous staining was a feature of only a few,specific cancer types including endometrial carcinomas (see FIGS. 39A-D)and malignant melanomas (88% and 92%, respectively), metaplastic breastcarcinomas, chondrosarcomas and liposarcomas (both 100%). See Tables 30and 33.

Simultaneous expression of PD-L1 in tumor cells and presence of PD-1+TILs was frequently observed in kidney cancer (33%), ovarian cancer(36%), NSCLC (43%), TNBC (45%), dedifferentiated liposarcomas (50%),bladder cancer (55%), malignant melanomas (58%), endometrial cancer(79%), but was infrequent in other cancer types in our cohort, e.g., 0%in liver cancer and Merkel cell carcinoma, 4% microsatellite-stable(MSS) colon cancer, 5% prostate cancer, 8% liver cancer, 9% pancreaticcancer, and 13% in luminal A breast cancer. See Table 30.

Association of PD-1 and PD-L1 Expression with Genotypic and PhenotypicCharacteristics of the Tumors

In the sample set used in this Example, expression of PD-1+ TILs wasassociated with an increasing number of mutations in tumor cells(p=0.029, Fisher's exact test) whereas PD-L1 status showed the oppositeassociation (p=0.004, Fisher's exact test). Consequently, co-presence ofPD-1+ TILs and cancer cells expressing PD-L1 showed no association withoverall mutational status (p=0.67, Fisher's exact test).

In breast cancer PD-1+ TILs were significantly more common in TNBC thanin luminal-type tumors (70% vs. 25-44%, p<0.001, Chi-square test). SeeTable 31. Similarly, PD-L1 expression was the highest in TNBC ascompared to other subtypes (59% vs. 33% in luminal tumors, p=0.017).Among TNBC, 9 cases were metaplastic breast carcinomas and all werepositive for PD-L1. Consequently, co-expression of PD-1+ TIL/cancercells PD-L1+ was the highest in the TNBC subgroup (45% vs. 13-17%non-TNBC, p=0.001, Chi-Square test). Similarly, TP53 mutated breastcancers exhibited significantly higher PD-1 TIL positivity compared withbreast cancers that harbored other mutations (e.g. PIK3CA mutations) orbreast tumors without mutations (42% vs. 10%, p=0.002, Chi-square test).In contrast, PD-L1+ did not correlate with any of the detected mutationsin breast cancer.

In the colon cancer cohort, MSI-H tumors exhibited a significantlyhigher rate of positivity for PD-1+ TILs than MSS colon cancers (77% vs.39%, p=0.002, Fisher's exact test). See Table 32. Also, the proportionof PD-L1+ cancers was significantly higher in MSI-H than in the MSScolon cancers (38% vs. 13%, p=0.02, Fisher's exact test). Of note, MSI-Hcases were predominantly stage I and II (75%) whereas the majority ofthe MSS cases were at advanced stage (III and IV, 93%) (p<0.001). BothPD-1 and PD-L1 positivity significantly decreased with the tumor stagein CRC (p=0.021 and 0.031, respectively).

In NSCLC, PD-1+ TILs and PD-L1 expressing tumor cells were seen in 18/42cases (43%) of which 8 cases lacked other biological targets (such asactivating EGFR mutations, HER2, cMET, ALK or ROS1 rearrangements).

Discussion

Recent clinical trials have demonstrated that blocking of the PD-1/PD-L1pathway induces an objective and durable remission in patients withadvanced solid tumors (2-6). The efficacy of these agents has beenprimarily linked to the expression of PD-L1 in the tumor cells and PD-1on activated T lymphocytes (12-14). Expression of both markers hasalready been explored in several human malignancies, particularly inrenal cell carcinomas, malignant melanoma and NSCLC (13-15). Our PD-L1results for these three cancer types are comparable with the dataprovided by Taube et al (13). Consistent with a previous report byVanderstraeten et al. (16), endometrial cancer appears to be abundantlyenriched with both PD-1 and PD-L1.

The broad array of tumors screened for this study also allowed theassessment of PD-1/PD-L1 expression in several less common cancer types.Our study revealed a low expression of both PD-1 and PD-L1 in severalhighly aggressive tumors including Merkel cell carcinoma, hepatocellularand pancreatic carcinoma. In contrast, PD-L1 expression was particularlyhigh in dedifferentiated liposarcomas, which is in line with a recentreport by Kim et al. (17). We also found PD-L1 positivity inchondrosarcomas and extraskeletal myxoid chondrosarcomas. Furthermore,PD-1 and PD-L1 positivity was observed in cancers of unknown primary, agroup of cancers with particularly difficult treatment decisions.

Marked variations in PD-1/PD-L1 positivity have also been observedwithin general histologic types, but subtype analysis revealedsignificant correlations. For example, PD-1/PD-L1 were differentlyexpressed in molecular subtypes of breast (TNBC vs. non-TNBC) and coloncancer (MSI-H vs. MSS cases) providing an indication for potentialbenefit of targeted immunotherapy in aggressive subtypes of breast andcolon cancers for which no targeted therapy is currently available. Wefound PD-L1 expression to be the highest in TNBC (59%) whereas a recentstudy that reported the highest frequency (34%) in HER2-positive breastcancers (18). The difference may be due to the cohorts analyzed. Ourcohort was enriched (8%) for rare metaplastic TNBC, which were all PD-L1positive whereas we analyzed only 5 HER2-positive breast cases. Of note,TP53 mutated breast carcinomas exhibited significantly higher PD-1expression in comparison with breast carcinomas harboring other types ofmutations. High PD-1+ TILs had been recently associated with a moreaggressive phenotype and poorer outcome in operable breast cancers (19).

Upon interferon-gamma (IFN-γ) stimulation, PD-L1 is expressed onT-cells, NK-cells, macrophages and vascular endothelial cells, allpresent in tumor microenvironment and detected in nearly all of ourcases. Some immunogenic tumors (e.g. MSI-H CRC) attract TILs whichproduce IFN-γ and could upregulate PD-L1 on tumor epithelial cells.IFN-γ receptor (IFN-γRa) expressed on tumor epithelial cells plays acritical role in tumor immunoediting (20), including acquisition of stemcell-like phenotype (21) and resistance to granule-mediated cytotoxicT-lymphocyte killing (22).

Our data for colon cancer also appear to differ from those reported byDroeser et al. who reported more frequent expression of PD-L1 in the MSSthan in MSI-H colon cancers (23). The discrepancy may be caused by thefact that tested MSI-H and MSS cases differed significantly in regardsto the tumor stage as the majority of MSI-H was at stage I and II whileMSS tumors were predominantly stage III and IV. Overall, the expressionof both PD-1 and PD-L1 in colon cancer inversely correlated with thetumor stage.

Another relevant finding in our study is that a substantial proportionof NSCLCs with PD-1/PD-L1 positivity were devoid of the most common andtargetable alterations (e.g. EGFR, HER2, cMET, ALK, ROS1). In contrastto previous studies, we did not find any association between PD-1/PD-L1expression and EGFR alterations in lung cancer (24, 25).

Without being bound by theory, low percentage of intra-tumoral PD-1+lymphocytes and PD-L1 cancer cells in certain solid tumors (see Tables30-33) may explain—in whole or in part—the observed lack of a benefitfrom therapies targeting this pathway. Also without being bound bytheory, these data are consistent with the idea that PD-1 lymphocytesthat are in direct contact with (PD-L1 expressing cancer cell) are mostrelevant for response to PD-1/PD-L1 targeted therapies. Thus,cell-to-cell contact (PD-1 lymphocytes with PD-L1 cancer cell) may beused as a potential biomarker of response. Such interactions in a tumormay indicate the efficacy of PD-1/PD-L1 pathway modulators. Dual IHCand/or flow cytometry may provide such a signal. See, e.g., Segal andStephany, The Measurement of Specific Cell:Cell Interactions byDual-Parameter Flow Cytometry, Cytometry 5:169-181 (1984).

In summary, our survey demonstrated expression of two potentiallytargetable immune checkpoint proteins (PD-1/PD-L1) in a substantialproportion of solid tumors including some aggressive subtypes that lacktargeted treatment modalities. In some other tumor types, expression ofthe immune checkpoint proteins was rare. Taken together, these dataindicate that molecular profiling can be used to assess likely benefitof PD-1 and PD-L1 therapies across a broad variety of tumor types.

LITERATURE

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Modulation of the tumor cell phenotype by IFN-γ results in    resistance of uveal melanoma cells to granule-mediated lysis by    cytotoxic lymphocytes. J Immunol 2008; 180:3766-74.-   23. Droeser R A, Hirt C, Viehl C T, Frey D M, Nebiker C, Huber X, et    al. Clinical impact of programmed cell death ligant 1 expression in    colorectal cancer. Eur J Cancer 2013; 49:2233-42-   24. Yang C Y, Lin M W, Chang Y L, Wu C T, Yang P C. Programmed cell    death-ligand 1 expression in surgically resected stage I pulmonary    adenocarcinoma and its correlation with driver mutations and    clinical outcomes. Eur J Cancer 2014; 50:1361-9.-   25. Akbay E A, Koyama S, Carretero J, Altabef A, Tchaicha J H,    Christensen C L, et al. Activation of the PD-1 pathway contributes    to immune escape in EGFR-driven lung tumors. Cancer Discov 2013;    3:1355-63.

Example 11: Predicative Biomarker Profiling of 2000 Sarcomas

Sarcomas are a heterogeneous group of tumors with more than 50 subtypes.First line chemotherapy such as doxorubicin and ifosfamide yield limitedsurvival benefit. In this Example, molecular profiling of the inventionwas used to guide new therapeutic options for sarcoma.

A total of 2047 sarcomas (including soft tissue sarcomas—leiomyosarcoma,fibrosarcoma, rhabdomyosarcoma, liposarcoma, angiosarcoma, synovialsarcoma, malignant peripheral nerve sheath tumor; organ specificsarcomas—angiosarcoma of the breast, osteosarcoma and chondrosarcoma,Ewing sarcoma of bone) were profiled using comprehensive molecularprofiling as described herein, including biomarker assessment using IHC,Sanger sequencing, Next Generation sequencing, and FISH/CISH. IHC datawas available for 1968 samples, FISH/CISH data for 1048 samples, andsequencing data for 261 samples. Data for all platforms was availablefor 256 samples. 713 samples were known to be from a metastatic site,the median patient age was 55 (range: 1-92), and 62% of the patientswere female. Tumor types are displayed in Table 34.

TABLE 34 Histological types Alveolar soft part sarcoma (ASPS) 14Angiosarcoma (11 = breast) 65 Chondrosarcoma 47 Chordoma 12 Clear cellsarcoma 14 Desmoplastic small round cell tumor (DSRCT) 31 Epithelioidhemangioendothelioma (EHE) 12 Epithelioid sarcoma 14 Endometrial stromalsarcoma (ESS) 72 Ewing sarcoma 66 Fibromatosis 36 Fibrosarcoma 61 Giantcell tumour 11 Leiomyosarcoma (LMS; 355 are uterine) 630 Liposarcoma 168Malignant fibrous histiocytoma (MFH/UPS) 145 Malignant peripheral nervesheath tumor (MPNST) 31 Osteosarcoma 84 Perivascular epithelioid celltumor (PEComa) 16 Rhabdomyosarcoma 67 Solitary fibrous tumor (SFT) 36Synovial sarcoma 61 fibromyxoid sarcoma 9 Fibrous hamartoma of infancy 1hereditary leiomyomatosis 1 angiomyolipoma 1 angiomyxoma 1 Atypicalspindle cell lesion (with fibrohistiocytic 1 differentiation)chondroblastoma 1 dendritic cell sarcoma 3 Granular cell tumor 6 Highgrade myxoid sarcoma 1 high-grade myoepithelial carcinoma 1 Hyalinizingfibroblastic sarcoma 1 Inflammatory myofibroblastic sarcoma 1Interdigitating Dendritic Cell Tumor 1 Intimal sarcoma 3 leiomyoma 2lymphangitic sarcomatosis 1 malignant glomus tumor 1 Malignantmyoepithelioma 1 melanocytic neoplasm 1 mesenchymal neoplasm 3Mesenteric glomangioma 1 Metastatic histocytoid neoplasm 1myoepithelioma 1 myxoid sarcoma 4 myxoid stromal 1 neurilemmoma 1phyllodes 12 rhabdoid 4 Round cell 2 Sarcoma, NOS 204 sarcomatousmesothelioma 1 schwannoma 4 Spindle and round cell sarcoma 1 Spindlecell 75 Spinocellular mesenchymal tumor 1

Overall IHC results are displayed in Table 35. About 50% or the sarcomasoverexpressed TOPO2a, and there was PTEN loss in 43%. EGFR wasoverexpressed in 36% in a wide variety of sarcomas (liposarcoma, UPS,LMS). CKIT, HER2, and cMet were overexpressed at low levels overall.

TABLE 35 Overall Immunohistochemistry Results Protein Below ThresholdAbove Threshold Total % Above AR 1653 256 1909 13.4 BCRP 228 224 45249.6 cKIT 1333 60 1393 4.3 cMET 528 33 561 5.9 EGFR 125 70 195 35.9 ER1583 337 1920 17.6 ERCC1 881 589 1470 40.1 Her2 1949 1 1950 0.1 MGMT1221 641 1862 34.4 MRP1 412 867 1279 67.8 PDGFR 443 128 571 22.4 PGP1414 223 1637 13.6 PR 1508 404 1912 21.1 PTEN 816 1091 1907 57.2 RRM11161 548 1709 32.1 SPARC 1264 704 1968 35.8 TLE3 441 142 583 24.4 TOP2A822 844 1666 50.7 TOPO1 884 767 1651 46.5 TS 1321 380 1701 22.3 TUBB3186 106 292 36.3

Table 36 shows a selection of IHC results by histology. For MGMT andRRM1, underexpression potentially confers sensitivity to an agent.Therefore, low MGMT expression in the majority of fibromatosis and LMSpotentially confers sensitivity to alkylating agents such astemozolomide. Low RRM1 expression the ASPS tumors and the majority offibromatosis and liposarcoma potentially confers sensitivity togemcitiabine. On the other hand, overexpression of SPARC, as observed in50-70% of angiosarcoma, chondrosarcoma and EHE and osteosarcoma,indicates likely benefit of nab-paclitaxel. TOPO2A overexpression, whichindicates likely benefit of anthracyclines, was seen in approximately60% of angiosarcoma, LMS and UPS.

TABLE 36 Immunohistochemistry Results by Histology (% overexpressing)Histology N MGMT* RRM1* SPARC TOPO2A ASPS 14 21.4 0.0 14.3 9.1Angiosarcoma 64 48.4 39.0 53.1 63.8 Chondrosarcoma 47 70.2 20.5 51.114.3 EHE 12 16.7 27.3 66.7 0.0 Epithelioid sarcoma 14 46.2 38.5 30.815.4 Fibromatosis 34 3.2 10.7 48.5 0.0 LMS 610 22.8 33.3 30.7 62.0Liposarcoma 158 40.0 15.8 35.4 31.4 MFH/UPS 140 24.8 25.6 36.6 63.9Osteosarcoma 80 29.1 38.2 47.6 48.6 *Expression of the biomarker belowthe threshold is considered predictive of a positive response to therapy

Table 37 shows another selection of IHC results by histology. ARoverexpression was noted in 20-40% of chondrosarcoma, DSRCT, ESS andLMS. cKIT overexpression was noted in 29% angiosarcoma, 37% Ewingsarcoma. cMET overexpression 25% Ewing sarcoma. ER alpah overexpressionas expected was seen in 20-45% of ESS and LMS. 60% in uterine and 21% inextrauterine. There was also expression above average in PEComa. PTENloss was seen in 60-80% of epithelioid sarcoma, osteosarcoma andrhabdomyosarcoma. 41% of LMS had PTEN loss the same regardless ofanatomical site.

TABLE 37 Immunohistochemistry Results by Histology (% overexpressing)Histology N AR cKIT cMET ERα PDGFRA PTEN* Angiosarcoma 64 0.0 28.6 9.50.0 46.7 50.8 Clear cell sarcoma 12 0.0 0.0 50.0 0.0 50.0 63.6Chondrosarcoma 47 23.9 4.3 9.1 0.0 40.0 63.8 DSRCT 30 40.0 19.0 11.1 0.07.7 50.0 EHE 12 8.3 0.0 0.0 0.0 33.3 75.0 Epithelioid sarcoma 14 0.0 0.00.0 0.0 25.0 23.1 ESS 71 28.2 1.8 5.9 46.5 40.0 78.9 Ewing sarcoma 633.6 37.3 25.0 5.4 31.8 41.7 LMS 610 22.4 1.1 3.9 43.2 15.4 59.2Osteosarcoma 80 2.6 0.0 0.0 0.0 27.8 29.6 PEComa 16 12.5 0.0 0.0 25.00.0 81.3 Rhabdomyosarcoma 64 8.5 9.3 15.0 3.3 17.4 41.0 *Expression ofthe biomarker below the threshold is considered predictive of a positiveresponse to therapy

33 additional patients had tumor submitted for PD1 and PDL1 analysis. Asshown in Table 38, all of the 20 liposarcomas and 9 chondrosarcomasexpressed PDL1 in at least 5% of cells with at least a stainingintensity of 2.

TABLE 38 Immunohistochemistry Results for PD-1/PD-L1 expression PD-1PD-L1 Concurrent N expression/ (tumor PD-1 and PD-L1 Sarcoma subtype(33) hpf (TILs) cells) expression Liposarcoma 20 45% 100% 45%Chondrosarcoma 9 11% 100% 11% Extraskeletal myxoid 3  0%  67%  0%chondrosarcoma Uterine sarcoma 1  0% 100%  0%

Overall FISH/CISH results are displayed in Table 39. There was a lowlevel of TOPO2a amplification, but amplification of EGFR was observed in17% of the cases. Although the level of HER2 amplification was lowoverall, this was amplification was concentrated in 3-8% of ESS and LMS,the latter more commonly found in the extrauterine LMS: 1.5% in uterineLMS and 4% in extrauterine LMS. EGFR amplification was observed ingreater than 5% of Chondrosarcoma, ESS and Ewing sarcoma, greater than10% of Fibrosarcoma, Liposarcoma and Rhabdomyosarcoma, and greater than20% of LMS, MPNST, Osteosarcoma and UPS.

TABLE 39 Overall In situ Hybridization (ISH) Results Assay Total NormalAmplified % Amplified cMET 431 414 17 3.9 cMYC 18 17 1 5.6 EGFR 1048 872176 16.8 HER2 573 565 8 1.4 TOP2A 107 105 2 1.9

PTEN loss was observed in up to 80% in several different histopathologytypes including angiosarcoma, Kaposi's sarcoma, LMS, liposarcoma,rhabdomyosarcoma, Ewing's sarcoma, Osteosarcoma, chondrosarcoma andothers. Overexpression of TOPO2 and TOPO1 proteins were observed in morethan 50% of angiosarcomas, fibrosarcomas, leiomyosarcomas,rhabdomyosarcomas, malignant fibrous histiocytomas, malignant peripheralnerve sheath tumors, desmoplastic small round cell tumors, synovialsarcomas, and hemangiopericytoma. Low MGMT expression was observed in75% of osteosarcoma. Absence or low TS expression was seen in Kaposisarcoma, leiomyosarcomas, hemangiopericytomas and liposarcomas. Steroidhormone receptor overexpression was observed in Ewing sarcomas (52%) anddesmoplastic small round cell tumors (44%), followed byrhabdomyosarcomas (36%) and leiomyosarcomas (25%). cMET by FISH showedamplification in 17% of leiomyosarcoma tested, while EGFR FISH showed >4copies in more than 30% of malignant fibrous histiocytomas and malignantperipheral nerve sheath tumors tested.

Of 261 patients tested using NGS with the panel in Table 8, 156 had nomutations (60%) and the rest had 123 gene aberrations detected in 25genes. Some of the most common mutations in the overall population areshown in Table 40. Most of the mutations were at low levels in theentire population, 22.4% had p53 mutations. In this population, only 1mutant was found each of ABL1, AKT1, AKT1, FGFR2, FLT3, GNA11, KDR,MLH1, SMARCB1 and SMO. And no mutations were detected in ALK, CDH1,CSF1R, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, GNAQ, GNAS, HRAS, JAK2, MPL,NOTCH1, NPM1, PDGFRA, PTPN11, SMAD4 and VHL.

TABLE 40 Next Generation Sequencing Gene Total Tested WildType Mutated %Mutated APC 261 254 7 2.7 ATM 258 252 6 2.3 BRAF 542 534 8 1.5 cKIT 394389 5 1.3 cMET 260 254 6 2.3 CTNNB1 261 255 6 2.3 IDH1 261 257 4 1.5JAK3 260 257 3 1.2 KRAS 1473 1454 19 1.3 NRAS 365 362 3 0.8 PIK3CA 333323 10 3 PTEN 249 241 8 3.2 RB1 258 252 6 2.3 STK11 247 243 4 1.6 TP53254 197 57 22.4

Some of the mutations occurring at higher frequencies in varioushistologies are shown in Table 41, including some known mutation such asIDH1 in chondrosaroma or cKIT in synovial sarcoma, and others such asBRAF in angio, PIK3CA in a variety of sarcomas. The data include bothSanger and NGS results. Table 42 displays similar data for raresarcomas. The data revealed known mutations such as CTNNB1 infibromatosis, and also BRAF in MPSNT and PIK3CA and PTEN infibrosarcoma. Other histologies had either no mutations detected otherthan TP53.

TABLE 41 % mutated by histology Angio LMS Synovial Histology (all)Chondro (all) Liposarcoma UPS sarcoma APC 13.3 0 2.3 0 0 0 ATM 6.7 0 03.3 0 10 BRAF 10 0 0 2.1 2.4 0 cKIT 0 0 0 0 3.1 11.8 cMET 6.7 0 4.5 3.30 0 CTNNB1 0 0 0 0 0 0 IDH1 0 25 0 0 4.2 0 JAK3 0 0 0 3.3 0 0 KRAS 5.8 00 0 2.7 0 NRAS 13.3 0 0 0 0 0 PIK3CA 0 0 1.6 5.6 3.8 0 PTEN 6.7 16.7 7.13.6 0 0 RB1 0 0 7 0 4.2 0 STK11 0 0 2.5 3.7 0 0 TP53 26.7 25 41.5 13.334.8 0

TABLE 42 % mutated by histology, rare sarcomas Fibro- Fibro- GiantHistology ESS matosis sarcoma MPNST cell tumor Rhabdo APC NT 14.3 0 033.3 0 ATM NT 0 14.3 0 0 0 BRAF 0 0 0 7.1 0 4.2 cKIT 0 0 0 0 0 0 cMET NT0 0 0 0 0 CTNNB1 NT 85.7 0 0 0 0 IDH1 NT 0 0 0 0 0 JAK3 NT 0 0 0 0 0KRAS   6.1 0 6.1 3.8 20 2.4 NRAS 0 0 0 0 0 0 PIK3CA 0 0 6.7 0 0 10 PTENNT 0 16.7 0 0 0 RB1 NT 0 0 0 0 0 STK11 NT 14.3 0 0 0 0 TP53 NT 0 28.611.1 0 11.1

Some specific mutations observed are shown in Table 43. These mutationswere observed most frequently excluding p53. BRAF v600E was the mostcommon BRAF mutation. PTEN alterations were mostly frame shift mutationswith some missence mutations.

TABLE 43 Specific mutations Gene Alteration Exon Frequency Histology (N)BRAF V600E 15 5 Liposarcoma (1), angiosarcoma (1), MPNST (1), other (2)cKIT T67S 2 2 UPS (1), synovial sarcoma (1) cMET T1010I 14 3Angiosarcoma (1), LMS (1), liposarcoma (1) IDH1 R132C 3 4 Chondrosarcoma(3), UPS (1) KRAS G12C 5 2 UPS (1), angiosarcoma (2), giant cell tumor(2) KRAS G12V 2 4 Fibrosarcoma (1), UPS (1), MPNST (1), other (2) PIK3CAH1047R 20 3 Liposarcoma (1), fibrosarcoma (1), other (1) PIK3CA E545K 93 LMS (1), liposarcoma (1), other (1)

In sum, the following mutations other than TP53 were detected withfrequency ≥5%: 1) Synovial sarcoma and ATM, cKIT; 2) Angiosarcoma andBRAF, APC, NRAS, ATM, cMET, KRAS, PTEN; 3) Chondrosarcoma and IDH1,PTEN; 4) Liposarcoma and PIK3CA; and 5) LMS and PTEN, RB1. These datasuggest targeted therapy such as against cKit in synovial and againstBRAF in angiosarcoma.

Association of p53 mutations with other alterations and PIK3CA mutationsand other alterations were investigated. See Tables 44-45. 82% ofsamples were both TOPO2 positive by IHC and p53 mutated. These datasuggest that P53 mutations may serve as a biomarker of sensitivity toanthracyclines. One patient had PTEN loss and PIK3CA mutation, which isnot previously described in the literature. PIK3CA and PTEN mutationswere mutually exclusive in the tumors tested.

TABLE 44 Coincidence of alterations with TP53 mutation PTEN Loss TOPO2APTEN cMET IDH CTNNB1 APC KRAS IHC IHC+ MT MT MT MT MT MT TP53wt 24/19798/182 5/192 2/201 1/202 6/202 5/202 6/201 (12.2%) (53.8%) (2.6%) (1.0%)(0.5%) (3.0%) (2.5%) (3.0%) TP53 10/51  41/50  3/52  4/52  3/52  0/52 2/52  0/52  mutated (19.6%)  (82%) (2.6%) (7.7%) (5.8%) (0) (3.8%) (0) Pvalue   0.17 0.0003 0.37  0.03  0.03  0.35 0.63  0.35

TABLE 45 Coincidence of alterations with TP53 mutation TP53 MT PTEN LossIHC TOPO2A IHC+ PTEN MT PIK3CA mutated 3/7 (42.9%; 1LMS, 1 lipo)  1/10(10.0%)   7/8 (87.5%) 0/6 (0)   PIK3CA WT 54/243 (22.2%) 39/316 (12.3%)136/229 (59.4%) 2/240 (0.8%) P value 0.40 1.0 0.15 1.0

26. Distinct biomarker expression and molecular phenotypes identifiedtherapeutic strategies not otherwise considered in the treatment ofsarcoma. Alterations with therapeutic implications were found in 99% ofsarcomas. For example, PTEN protein expression and EGFRpolysomy/amplification have been associated with potential benefit toEGFR pathway targeted therapy. Overexpression of TOPO2 and Topo 1 canfine tune the use of anthracyclines and irinotecan in significantnumbers of patients. The overexpression of TOPO2 was observed inapproximately 50% of sarcomas, without concomitant amplification. Thiswas most commonly observed in angiosarcoma, LMS, and UPS. These datasuggest sensitivity to anthracyclines, especially in relation to TP53status in a tumor. SPARC is overexpressed in angiosarcoma,chondrosarcoma, EHE and osteosarcoma, which suggests sensitivity tonab-paclitaxel in addition to the current taxane therapy. PTEN loss wasseen in 80% of sarcomas without associated mutations and the use ofPI3kinase inhibitors in this subset of patients may be beneficial. PDL1was expressed in all of the liposarcomas (mostly dedifferentiated) andchondrosarcomas, this indicating potential benefit from the use of thenew immune checkpoint inhibitors (anti-PD-1 or anti-PD-L1 therapy). Highlevel of steroid hormone receptor expression uncovers the potential touse anti-steroid hormone therapy in some rare sarcomas. Low MGMTexpression suggests potential benefit from radiation therapy andtemozolomide, while tumors with low TS expression may benefit from theuse of fluorouracil based therapies. The presence of activatingmutations BRAF V600E and PIK3CA E545K or H1047L provide for highlyspecific targeted inhibitors. Trials of agents like mTOR and PI3Kinhibitors could benefit from designs in which patient selection isbased on PTEN loss or PIK3CA mutations instead of sarcoma histology.Overall, molecular profiling through protein expression, gene copyvariations and mutations identified alterations in 99% of sarcomasamples which may guide the most beneficial treatment options.

Example 12: Identification of Actionable Targets in Rare Cancers Using aMultiplatform Molecular Analysis

Chordomas are a rare cancer. Limited biomarker data exists toprognosticate outcome or predict response to therapy. This Exampleexplores the utility of multiplatform tumor profiling, which usesimmunohistochemistry (IHC) and next generation sequencing (NGS) asdescribed herein to identify druggable targets in patients withchordoma.

All tissues were internally reviewed by a pathologist.Immunohistochemistry (IHC) was performed on AR, BCRP, cKIT, cMET, EGFR,ER, ERCC1, HER2, MGMT, MRP1, PD-1, PD-L1, PDGFR, PGP, PR, PTEN, RRM1SPARC, TLE3, TOP2A, TOPO1, TS and TUBB3. In situ hybridization(fluorescence or chromogenic) was performed on EGFR, HER2, cMET andTOP2A. Sequencing (Sanger or NGS) was performed on the genes listed inTable 8.

31 chordoma patients were profiled, of which 12 had metastatic disease.The median age was 58 years old; 58% of patients were male.Overexpression of EGFR and TOPO1 were identified in 50% and 54% ofcases, while Phosphatase and tensin homolog (PTEN), thymidylate synthase(TS), ribonucleotide reductase M1 (RRM1), and O-6-methylguanine-DNAmethyltransferase (MGMT) expression was absent in 15/25, 22/26, 21/26and 8/23 tumors, respectively. A pathogenic point mutation in PIK3CA(Q546R) was detected in 1 of 12 tumors tested whereas no mutations wereidentified in the other 46 genes tested by sequence analysis. No changesin copy number were identified using ISH. Notably, PD-1 tumorinfiltrating lymphocytes (TILs) and PD-L1 were seen in 20% and 60% ofcases tested, respectively.

Biomarker analysis indicates that chordomas might be amenable tochemotherapy with 5-fluorouracil, gemcitabine, or temozolamide due toabsence of TS, RRM1, and MGMT expression, respectively. Targeting thePI3 kinase pathway is supported by the high loss of PTEN and the PIK3CAmutation. Additionally, immunotherapies, e.g., anti-PD1 therapy, mightbe of utility in this rare cancer based on the 60% of cases in whichtumor infiltrating lymphocytes were identified.

Similar analysis as above was performed for a cohort of rare adrenaltumors, including 142 tumors of the adrenal cortex, 33 of the adrenalmedulla, 2 paraganglia and 7 soft tissues of the abdomen (specifically,neuroblastoma/ganglioneuroblastoma of the periadrenal soft tissue). 49%of the tumors were recurrent and 8.6% were metastatic. The average agewas 48 (range=20-86) and 59% were female. Of the 142 adrenal corticaltumors, 137 were adrenal cortical carcinoma, one was a large cellcarcinoma, one was a carcinosarcoma, one was a neuroendocrine tumor, onewas a malignant neoplasm, and one was unspecified.

Results of protein expression analysis are shown in Table 46. Results ofamplification/rearrangements analysis are shown in Table 47. Mutationsby detected by next generation sequencing are shown in Table 48. Nomutations were observed in this cohort in ABL1, ALK, BRAF, BRCA1, CDH1,CSF1R, ERBB2, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS,IDH1, JAK2, KRAS, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN,PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, STK11 or VHL.

TABLE 46 Immunohistochemistry Results for Adrenal Cortical tumorsExpression observed Total tested % AR 10 135 7.4 BCRP 26 34 76.5 c-kit 682 7.3 cMET 1 56 1.8 EGFR 10 30 33.3 ER 3 135 2.2 ERCC1^($) 33 76 43.4HER2 0 132 0 MGMT^($) 38 118 32.2 MRP1 57 59 96.6 PD-1 4 11 36.4 PD-L1 411 36.4 PDGFR 4 56 7.1 PGP^($) 62 111 55.9 PR 79 134 59 PTEN^($) 53 12442.7 RRM1^($) 47 115 40.9 SPARC 65 139 46.8 TLE3 5 58 8.6 TOP2A 51 11544.3 TOPO1 50 108 46.3 TS^($) 42 115 36.5 TUBB3^($) 5 43 11.6^($)Expression of the biomarker below the threshold is consideredpredictive of response to therapy.

TABLE 47 ISH Results for Adrenal Cortical tumors Alterations Totaltested % cMET 1 42 2.4 EGFR 5 46 10.9 HER2 1 66 1.5

TABLE 48 NGS Results for Adrenal Cortical tumors Mutation Detected Totaltested % AKT1 1 32 3.1 APC 1 33 3 ATM 1 32 3.1 BRCA2 1 7 14.3 c-KIT 1 323.1 cMET 1 33 3 CTNNB1 11 33 33.3 EGFR 1 35 2.9 ERBB4 1 33 3 JAK3 1 323.1 KDR 1 32 3.1 TP53 15 32 46.9

Of note, over a third of the tumors tested expressed both PD-1 and PD-L1(see Table 51). These data suggest utility of targeted immunotherapies,e.g., anti-PD1 therapy or anti-PD-L1 therapy, to treat these rarecancers.

Example 13: Molecular Profiling of Non-Urothelial Bladder Cancer:Adenocarcinoma and Squamous Cell Carcinoma

Background: Adenocarcinoma (ADA) and squamous cell carcinoma (SCC) arerare and often aggressive histologic subtypes of bladder cancer. Foradvanced disease, no clear standard therapies exist and NCCN guidelinessuggest only fluorouracil, cisplatin, paclitaxel and ifosfamide aspossible options. Thus, novel therapies based on underlying tumorbiology are needed. The purpose of this study was to identify potentialtargets and therapeutic options for these histologic subtypes, utilizingmultiplatform tumor profiling.

Methods: 49 ADA and 24 SCC specimens were tested via a multiplatformmolecular profiling service of the invention consisting of genesequencing (Sanger or next generation sequencing [NGS]), geneamplification (CISH or FISH), and protein expression(immunohistochemistry [IHC]). Tissue from a metastatic site wassubmitted in 52% of the cases.

Results: Both ADA and SCC exhibited high rates of TP53 aberrations(82.4% and 72.7%, respectively). Sequencing revealed mutations in BRCA2(14.3%), SMAD4 (12.5%), PTEN (11.8%), KRAS (8.7%), NRAS (5.6%), and KIT(5.3%) in ADA. In addition, PIK3CA (21.4%), HRAS (18.2%), BRCA1 (16.7%),BRCA2 (16.7%), and FBXW7 (9.1%) mutations were detected in SCC.Amplification in EGFR (27.3%) and ERBB2/HER2 (16.7%) were found in ACA.Meanwhile, only one ERBB2 (6.3%) amplification was found in SCC usingISH. MET was not amplified in either ACA or SCC. For both ACA and SCC,EGFR had the highest level of protein expression (100% and 85.7%,respectively). Of note, PD-1 (44.4% in both) and PD-L1 (11.1% and 22.2%in ACA and SCC, respectively) were expressed in both subtypes. Althoughdifferential rates of somatic alterations, amplification, and proteinexpression were found between ADA and SCC, only TLE3 was significant(19.2% versus 60.0%, respectively, p=0.0154).

Conclusion: Differential results in gene alteration, amplification, andprotein expression imply the potential utility of tumor profiling inguiding therapeutic decision-making in ADA and SCC of the bladder.Aberrations in the PIK3CA/AKT/mTOR pathway and alterations in TP53 inthese subtypes are similar to what has been reported in urothelialbladder cancer. Targeting the PD-1/PD-L1 axis is a therapeutic option.

Example 14: Comprehensive Profiling of Renal Medullary and CollectingDuct Carcinomas

Background: Renal medullary carcinoma (RMC) is an aggressive malignancyaffecting predominantly young African Americans with sickle cell trait(SCT) or disease (SCD), while a pathologically similar collecting ductcarcinoma (CDC) affects patients without sickle cell trait. Clinicalresponses to chemotherapy and IL-2 in RMC/CDC are poor and newtherapeutic options are needed.

Design: 9 patients with RMC (ages 13-58 y. o., all male) and 15 patients(ages 26-74 y. o., M:F=13:2) with collecting duct carcinoma (CDC) werestudied. Expression of PD-L1 was evaluated with 2 monoclonal antibodies(SP142 and SP263) and tumor infiltrating lymphocytes (TIL) wereevaluated for PD1 expression (MRQ-22 antibody) usingimmunohistochemistry (IHC). Additional studies included ALK proteinexpression (D5F3 antibody), gene translocation (break apart FISH), nextgeneration sequencing (NGS), and microsatellite instability (MSI).

Results: Cancer cell PD-L1 expression above the threshold (≥2+, ≥5%) wasseen in 7/9 RMC and 5/13 CDC cases. Concordance between 2 PD-L1antibodies was 94.4%. PD-1+ TIL were absent in 6/18 cases and variablypresent in 12/18 cases (from 1 to >15 TIL/40× power field). No MSI wasdetected in any of the cases tested (0/6). No case expressed ALKprotein, but one case of CDC showed ALK gene re-arrangement. Mutationswere identified in SMARCB1, FH, TP53 (3×), ATM, BRCA2, CHEK2 (2×), NF2(3×), SETD2, and CDKN2A. Mutations in VHL or KDR were not detected inthese patients. One patient with RMC (and SCT) achieved completeclinical remission after treatment with bevacizumab plus paclitaxel.

Conclusion: RMC and CDC strongly express PD-L1 in 12 of 22 cases,suggesting that these patients may benefit from targeting the PD-L1/PD1interaction. The absence of MSI in these cancers indicates a differentmechanism of PD-L1 upregulation from colorectal carcinomas. Consistentwith our previous study that showed frequent activation of(pseudo)hypoxia-induced pathways in RMCs (Human Pathology 2011;42:1979), we describe a case of RMC successfully treated with anti-VEGFtherapy.

Example 15: Caveolin-1: Beyond a Marker for Basal-Like Breast Cancers

Introduction: Caveolin-1 (Cav1) is associated with basal-liketriple-negative (ER−/PR−/Her2-) breast cancers (TNBC). Its biologicalcontribution to this subtype of breast cancer has not been fullyexplored and questions persist regarding the molecular role of Cav1 incarcinogenesis.

Experimental Procedures: 34 TNBC (17 Cav1+/17 Cav1−) patientsmolecularly-profiled according to the invention were evaluatedretrospectively for insight on the role Cav1 plays in TNBC. Thetranscriptome of 34 cases (samples analyzed contained ≥50% neoplasticcells), were profiled using Illumina's HumanHT-12 microarray (v4) (CarisLife Sciences, AZ). Data were normalized using mean normalizationprocedure. Differential expression analysis was performed using R'sLimma package. Pathway analysis was carried out using R's signalingpathway impact analysis (SPIA) package with 69 cancer, immunity, andcell signaling related KEGG pathways.

Results: Using the cutoff of two fold and adjusted p-value of 0.05, weidentified 954 genes differentially expressed between Cav1+ and Cav1−TNBC patients. Included in these were 31 genes which were found to beup-regulated by over five-fold and 3 genes down-regulated by over fivefold in Cav1+ TNBC. Genes of notable interest for their role in cellsignaling, cell adhesion, tumor invasion and metastasis, included anup-regulation of TGFBR2, SPARC, integrins (ITGA11, ITGB5, ITGBL1), celladhesion proteins (LAMB3, COL5A3) and molecules which facilitate tumorinvasion (LAMB3, MMP1, MMP2, MMP9). In addition, genes found to bedown-regulated in Cav1+ patients, and notable for their roles inpromoting epithelial to mesenchymal transition (EMT) included Claudin 3(CLD3) and CA125/MUC16 (Mucin 16). We also detected an approximatelytwo-fold down-regulation of CDKN2A in Cav1+ patients. Using SPIA pathwayanalysis, 12 pathways were found to be differentially activated inCav1+vs. Cav1− TNBC patients. The most differentially activated pathwaywas the focal adhesion pathway (p=4.51E-18), PI3k-Akt signaling pathway(p=2.01E-6) and TGF-beta and MAPK signaling pathways (p=0.005, 0.014,respectively).

Conclusion: Differential gene expression patterns and pathway analysesprovides evidence for distinct profiles for gene expression between Cav1positive and negative TNBC. Cav1+ TNBC patients exhibit up-regulation ofgenes important for cell signaling, extracellular matrix remodeling andtumor invasion, and down-regulation of genes that may facilitate EMT andloss of cell cycle control. The focal adhesion pathway, as well asTGF-beta, PI3K and MAPK signaling pathways, were identified asdifferentially activated among Cav1+ and Cav1− TNBC. Taken together,this data supports the role of Cav1 positivity in identifying a subtypeof TNBC that may have a greater risk for invasion, metastasis andepithelial-mesenchymal-transition (EMT), and therefore a poorerprognosis, in need of aggressive treatments strategies.

Example 16: Mutations on the Homologous Recombination (HR) Pathway in 13Cancer Types

Background: HR pathway is important in DNA double strand break repair.Defects of HR promote carcinogenesis and are associated with selectivesensitivity to PARPi and DNA-damaging agents including platinum. We usednext-generation sequencing (NGS) to survey genes on the HR pathway in1029 tumors in 13 cancer types.

Method: NGS on ˜600 whole genes (see Tables 12-15) was performed usingformalin-fixed paraffin-embedded samples on the Illumina NextSeqplatform. All variants were detected with >99% confidence and with thesensitivity of 10%. Variants that are pathogenic or presumed pathogenicare counted as mutations.

Results: Table 49 summarizes mutation rates of 7 key genes (ATM, BRCA1,BRCA2, CHEK1, CHEK2, PALB2 and PTEN) included in this study. PTENmutations were seen in 6.3% of tumors, ATM in 5%, BRCA1 in 2%, BRCA2 in2%, PALB2 in 1%, CHEK2 in 1% and CHEK1 mutation is not seen in thecohort studied. Overall, 15% of tumors carry at least one mutation inany of the 7 genes, and the highest mutation rates were seen inendometrial (43%), GBM (34%) and gastric cancers (23%). The highestrates of ATM (9.7%), BRCA2 (6.5%) and PALB2 (6.5%) were seen in gastriccancer while the highest CHEK2 (5.6%), BRCA1 (7.3%) and PTEN (44%)mutations were seen in cholangiocarcinoma, ovarian and endometrialtumors, respectively.

Exceptional response was seen in a 53-year old patient with metastaticpoorly-differentiated adenocarcinoma of the stomach after 4 cycles ofFOLFOX without surgery, which included ongoing radiographic partialresponse and dramatic relief of symptoms. A nonsense mutation on PALB2(S326*) was found while the other 23 HRD genes were wild type; ERCC1 IHCshowed intact expression.

TABLE 49 Mutation rates of 7 key genes Biomarker Tumor type ATM BRCA1BRCA2 CHEK1 CHEK2 PALB2 PTEN Any of 7 Endometrial (N = 35) 0 0 0 0 2.9%3.0% 44.1% 42.9% GBM (N = 47) 2.1% 2.1% 0 0 0 0 30.4% 34.0% Gastric (N =31) 9.7% 0 6.5% 0 0 6.5% 0 22.6% Bladder (N = 38) 2.6% 0 5.4% 0 0 010.8% 18.4% Kidney (N = 41) 2.5% 0 0 0 5.0% 0 10.0% 17.1% Ovarian (N =82) 3.7% 7.3% 1.2% 0 1.2% 0 1.3% 14.6% Breast (N = 108) 4.6% 2.8% 1.9% 00.9% 1.0% 3.8% 13.9% Cholangiocarcinoma 2.8% 0 2.8% 0 5.6% 0 2.9% 13.9%(N = 36) CRC (N = 254) 6.3% 2.0% 1.6% 0 0.4% 0 4.0% 13.0% Pancreatic (N= 62) 4.8% 1.6% 3.2% 0 0 1.7% 3.3% 12.9% NSCLC (N = 234) 6.5% 0 0.9% 0 01.4% 2.6% 11.1% Neuroendocrine 2.9% 0 0 0 0 0 5.7% 8.6% (N = 35)Esophageal (N = 26) 3.8% 0 0 0 0 0 4.0% 7.7% Overall (N = 1029) 5.0%1.6% 1.6% 0 0.8% 0.8% 6.3% 15.2%

Conclusion: Mutation rates of at least 8 to 43% on the HR pathway arereported from 13 cancer types. This method can potentially identifyresponders to DNA-damaging agents including platinum.

Example 17: Clinico-Pathological and Molecular Features Associated withTP53 Mutation in 3457 Molecularly-Profiled Colorectal Cancers (CRCs)

Deregulation of the p53 tumor suppressor gene (TP53) is a key eventcontributing to transformation and aggressive metastatic features ofCRC. Patients with TP53 mutation are often resistant to therapy andcarry a poor prognosis. We investigated TP53 mutation in a cohort of3457 CRCs to identify molecular features specific to TP53-mutated CRCtumors. The 3457 CRC clinical samples were evaluated for tumor profilingas provided herein. Tests included Sanger or next generation sequencing(NGS), protein expression by immunohistochemistry (IHC) and geneamplification by in situ hybridization (ISH). TP53 mutation was observedin 2106 or 61% of CRCs analyzed. 2018 or 96% of these mutant TP53 tumorscarried one TP53 mutation, while 83 (4%) carried 2 mutations, 4 and 1tumors carried 3 and 4 mutations per tumor, respectively. Among the˜2200 mutations found in TP53, 37% were found at one of the six hotspotswithin the DNA binding domain (R175, G245, R248, R249, R273 and R282).Overall, 1554 (71%) were missense mutations, 367 (17%) nonsense, 209(9.5%) frameshift, 45 (2%) small in-dels, and 25 (1.1%) mutations thataffect splicing. In this cohort, TP53 mutation was more prevalent inmale patients (64% vs. 57%, P<0.0001) and was more likely to occur intumors that originated from the left colon (69%) as compared to theright colon (45%, p<0.0001). TP53 mutation rate was not correlated withpatient age, histology or whether the tumor sample was taken from theprimary or metastatic sites. When the molecular features of TP53-mutatedtumors were compared to those of wild-type TP53, mutated tumors carriedsignificantly higher Her2 IHC expression (2.5% vs. 1.0%, p=0.0039) andgene amplification (3.7% vs. 1.4%, p=0.0002), as well as higher MGMT(61% vs. 53%, p<0.0001) and TOPO2A expression (92% vs. 81%, p<0.0001).On the other hand, lower EGFR expression (57.4% vs. 70%, p<0.0001), PTENexpression (47.9% vs. 61%, p<0.0001), microsatellite instability (2.5%vs. 11.5%, p<0.0001), ERCC1 (18% vs. 24%, p<0.0001) and TS expression(31% vs. 38%, p<0.0001) were associated with TP53-mutated tumors.TP53-mutated CRCs carried higher rates of APC mutation (63% vs. 53%,p<0.0001), but lower rates of KRAS (46% vs. 54%, p<0.0001), PIK3CA(11.6% vs. 22%, p<0.0001), PTEN (2% vs. 5.2%, p<0.0001), GNAS (1% vs.8.3%, p<0.0001) and AKT1 (0.6% vs. 1.7%, p=0.0016) mutation. Thus, in acohort of 3457 molecularly profiled CRCs, TP53 mutation was moreprevalent in males and tumors that originated from the left colon.Distinct molecular features associated with TP53 mutation in CRCincluded lower frequency of PI3K/Akt/mTor pathway activation manifestedby significantly lower frequency of PIK3CA, PTEN and AKT1 mutations andhigher Her2 overexpression and amplification. These findings suggestdifferential presence of therapeutic targets in CRC tumors based on TP53mutation status.

Example 18: Expression of Class III Beta-Tubulin (TUBB3) in 3580Colorectal Cancers (CRCs) and Correlation with Clinico-Pathological andMolecular Features

Class III beta-tubulin plays crucial roles including maintenance of cellshape, intracellular transport, meiosis and mitosis. High expression ofTUBB3 has been shown to associate with poor prognosis and taxaneresistance in various cancer types. CRC is known to be generallyresistant to taxane therapy. We investigated expression of TUBB3expression in 3580 CRCs and made correlations with clinicopathologicaland molecular parameters. 3580 CRC samples were evaluated by tumorprofiling as provided herein. Tests included Sanger or next generationsequencing (NGS), protein expression by immunohistochemistry (IHC) andgene amplification by in situ hybridization (ISH). TUBB3 expression wasevaluated by immunohistochemistry (Ab: POLY, Covance) and expressionhigher than 2+, 30% was scored as positive. TUBB3 positive expressionwas observed in 37% (1320/3580) of the complete CRC cohort, specifically27% in mucinous histology (164/617) and 17% in signet ring histology(29/171). While the expression was not associated with average patientage (59 years old) or gender (53% vs. 50% male), TUBB3 was significantlymore frequently expressed in tumors that originated from the left colon(370/1016 or 36%) as compared to tumors from the right colon (235/790 or30%, p=0.003). In the 1847 tumors taken from the metastatic sites, 40%(746) overexpressed TUBB3 while in 1629 CRCs taken from the primarytumors, 34% (547) overexpressed TUBB3 (p<0.0001). Interestingly, intumors that overexpressed TUBB3, 65% also overexpressed cMET (828/1275)and 33% also overexpressed TLE3 (431/1299), as compared to 50% of cMETexpression (1096/2207, p<0.0001) and 23% of TLE3 (517/2225, p<0.0001) intumors that were negative for TUBB3. Microsatellite instability detectedby fragment analysis was more prevalent in the TUBB3-negative cohortthan the TUBB3-positive cohort (7.4% or 77/1046 vs. 2.9% or 16/558,p=0.0002). Similarly, while mutations on APC (62% vs. 56%, p=0.0003) andKRAS (55% vs. 46%, p<0.0001) were significantly more frequent inTUBB3-positive tumors, GNAS (2% vs. 5%, p<0.0001) and SMAD4 (10.1% vs.14.6%, p=0.0005) mutations were significantly more frequent in tumorsthat were negative for TUBB3 expression. Thus, high expression of TUBB3was found in 37% of CRCs, and was significantly associated with tumorsthat originated from the left colon and with tumors taken frommetastatic sites. Distinct biomarker features detected by IHC andsequencing suggest that TUBB3 expression carries theranostic value inthese patients.

Although preferred embodiments of the present invention have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A data processing system for identifying atherapeutic agent for an individual with a cancer, comprising: one ormore computers; one or more memory devices, wherein the one or morememory devices store: (i) a database having one or more fieldsstructuring data that includes: a. a reference value for each of aplurality of genes or gene products, and b. a listing of availabletherapeutic agents with efficacy known to be related to at least one ofa plurality of gene or gene products, and (i) instructions that, whenexecuted by the one or more computers, cause the one or more computersto perform operations, the operations comprising: generating, by the oneor more computers, an assay a plurality of first genes or gene productsin a biological sample from the individual to determine molecularprofile test values for the plurality of genes or gene products;determining, by the one or more computers and based on an analysis ofthe molecular profile test values and a corresponding reference valuefor each of the plurality of first genes or gene products accessed fromthe database, one or more therapeutic agents from the listing ofavailable therapeutic agents stored in the database; generating, by theone or more computers, data that, when processed by the one or morecomputers, causes the one or more computers to display data thatidentifies the one or more therapeutic agents.
 2. The data processingsystem of claim 1, wherein determining, by the one or more computers andbased on an analysis of the molecular profile test values and acorresponding reference value for each of the plurality of first genesor gene products accessed from the database, one or more therapeuticagents from the listing of available therapeutic agents stored in thedatabase comprises: determining, by the one or more computers a likelybenefit or lack benefit of the at least one therapeutic agent based on acomparison of the molecular profile test values and a correspondingreference value for each of the plurality of first genes or geneproducts accessed from the database.
 3. A method comprising: generating,by the one or more computers, an assay a plurality of first genes orgene products in a biological sample from the individual to determinemolecular profile test values for the plurality of genes or geneproducts; determining, by the one or more computers and based on ananalysis of the molecular profile test values and a correspondingreference value for each of the plurality of first genes or geneproducts accessed from a database, one or more therapeutic agents fromthe listing of available therapeutic agents stored in the database; andgenerating, by the one or more computers, data that, when processed bythe one or more computers, causes the one or more computers to displaydata that identifies the one or more therapeutic agents.
 4. The methodof claim 3, wherein determining, by the one or more computers and basedon an analysis of the molecular profile test values and a correspondingreference value for each of the plurality of first genes or geneproducts accessed from a database, one or more therapeutic agents fromthe listing of available therapeutic agents stored in the databaseincludes: determining, by the one or more computers a likely benefit orlack benefit of the at least one therapeutic agent based on a comparisonof the molecular profile test values and a corresponding reference valuefor each of the plurality of first genes or gene products accessed fromthe database.
 5. A computer readable medium storing instructions that,when executed by one or more computers, causes the one or more computersto perform operations that comprise: generating, by the one or morecomputers, an assay a plurality of first genes or gene products in abiological sample from the individual to determine molecular profiletest values for the plurality of genes or gene products; determining, bythe one or more computers and based on an analysis of the molecularprofile test values and a corresponding reference value for each of theplurality of first genes or gene products accessed from a database, oneor more therapeutic agents from the listing of available therapeuticagents stored in the database; and generating, by the one or morecomputers, data that, when processed by the one or more computers,causes the one or more computers to display data that identifies the oneor more therapeutic agents.
 6. The method of claim 5, whereindetermining, by the one or more computers and based on an analysis ofthe molecular profile test values and a corresponding reference valuefor each of the plurality of first genes or gene products accessed froma database, one or more therapeutic agents from the listing of availabletherapeutic agents stored in the database includes: determining, by theone or more computers a likely benefit or lack benefit of the at leastone therapeutic agent based on a comparison of the molecular profiletest values and a corresponding reference value for each of theplurality of first genes or gene products accessed from the database.